Importance of muscle selection for EMG signal analysis during upper limb rehabilitation of stroke patients.

Current work highlights the importance of muscle selection to evaluate paralysis and recovery level of stroke patients when comparing synergies of affected and non-affected side of the body. The proposed method allows the selection of important muscles that highly contribute to the specific movements according to the power and frequency distribution of the electromyographic signals.. Users participating performed steering-wheel-based therapy focused on upper limb rehabilitation. Final results show that with the appropriate muscles selection, it is possible to compute a Similarity Index between right and left arms (during symmetric motion) associated to the level of paralysis and potential recovery of a given subject.

Clarification of muscle synergy structure during standing-up motion of healthy young, elderly and post-stroke patients.

Standing-up motion is an important daily activity. It has been known that elderly and post-stroke patients have difficulty in performing standing-up motion. The standing-up motion is retrained by therapists to maximize independence of the elderly and post-stroke patients, but it is not clear how the elderly and post-stroke patients control their redundant muscles to achieve standing-up motion. This study employed the concept of muscle synergy to analyze how healthy young adults, healthy elderly people and post-stroke patients control their muscles. Experimental result verified that four muscle synergies can represent human standing-up motion. In addition, it indicated that the post-stroke patients shift the weights of muscle synergies to finish standing-up motion comparing to healthy subjects. Moreover, different muscle synergy structures were associated with the CoM and joint kinematics.

Tetramethylbenzidine method for monitoring the free available chlorine and microbicidal activity of chlorite-based sanitizers under organic-matter-rich environments.

UNLABELLED: Chlorine is a principal disinfectant for food and environmental sanitation. Monitoring of free available chlorine (FAC) is essential for ensuring the efficacy of food disinfection processes that rely on chlorine. N,N-diethyl-p-phenylenediamine (DPD) is commonly used for FAC monitoring. However, here, we show that upon contact with bovine serum albumin (BSA) or broiler carcasses, chlorite (HClO2 )-based sanitizers acquire a pink colour, which can interfere with measurement of oxidized DPD absorbance at 513-550 nm. Alternatively, the pink colour did not interfere with 3,3',5,5'-tetramethylbenzidine (TMB)-based FAC monitoring. The FAC levels of NaClO and weakly acidified chlorous acid water (WACAW) were first adjusted by the TMB method and the killing activity of these sanitizers towards methicillin-resistant Staphylococcus aureus (MRSA) and feline calicivirus (FCV) was compared in the presence or absence of 0·5% BSA. At 200 ppm FAC, NaClO lost its bactericidal activity against MRSA after 10-min incubation with 0·5% BSA. Meanwhile, under the same conditions WACAW reduced the number of bacteria to below the detection limit. Similar results were obtained with FCV, indicating that the chlorite-based WACAW sanitizer is relatively stable under organic-matter-rich conditions. Moreover, TMB is suitable for in situ FAC monitoring of chlorite-based sanitizers in food and environmental disinfection processes. SIGNIFICANCE AND IMPACT OF THE STUDY: For practical applications of chlorine in food processing, monitoring of FAC is critical to validate disinfection efficacy. In this study we found that chlorite-based sanitizers acquired a pink colour upon contact with BSA or broiler carcasses. This pink colour interfered with FAC monitoring by methods that measure oxidized N,N-diethyl-p-phenylenediamine absorbance between 513-550 nm. Alternatively, FAC levels of chlorite-based sanitizers could be monitored using the absorbance of 3,3',5,5'-tetramethylbenzidine at 650 nm, which does not overlap with the acquired pink colour. These data provide valuable information for safety management of disinfection processes that use chlorite-based sanitizers.

Field evaluation of a rapid diagnostic test to detect antibodies in human toxocariasis.

Human toxocariasis which is caused mainly by the larvae of Toxocara canis and Toxocara cati, is a worldwide zoonotic disease that can be a potentially serious human infection. The enzyme-linked immunosorbent assay (ELISA) using T. canis excretory-secretory (TES) antigens harvested from T. canis larvae is currently the serological test for confirming toxocariasis. An alternative to producing large amounts of Toxocara TES and improved diagnosis for toxocariasis is through the development of highly specific recombinant antigens such as the T. canis second stage larva excretory-secretory 30 kDa protein (recTES-30). The aim of this study was to evaluate the sensitivity and specificity of a rapid diagnostic kit (RDT, named as iToxocara kit) in comparison to recTES-30 ELISA in Serendah Orang Asli village in Selangor, Malaysia. A total of 133 subjects were included in the study. The overall prevalence rates by ELISA and RDT were 29.3% and 33.1%, respectively, with more positive cases detected in males than females. However, no association was found between toxocariasis and gender or age. The percentage sensitivity, specificity, positive predictive value and negative predictive value of RDT were 85.7%, 90.1%, 80% and 93.2%, respectively. The prevalence for toxocariasis in this population using both ELISA and RDT was 27.1% (36/133) and the K-concordance test suggested good agreement of the two tests with a Cohen's kappa of 0.722, P<0.01. In addition, the followed-up Spearman rank correlation showed a moderately high correlation at R=0.704 and P<0.01. In conclusion, the RDT kit was faster and easier to use than an ELISA and is useful for the laboratory diagnosis of hospitalized cases of toxocariasis.

Multiple drug combination of anti-diabetic agents as a predictor for poor clinical response to liraglutide.

AIM: Aim of the study was to retrospectively analyze the clinical parameters that contribute to the therapeutic outcome of GLP-1 analogues. METHODS: We enrolled 106 patients with type 2 diabetes mellitus (T2DM), treated with liraglutide (N.=69) or exenatide (N.=37) for longer than three months. The patients were divided into two groups: good responders and poor responders to GLP-1 analogues, based on pretreatment and post-treatment HbA1c levels. Good responders were those whose HbA1c level had decreased by 1% or more, or maintained at less than 7%. All other patients were categorized as poor responders. We used univariate and multivariate analyses to assess pretreatment parameters between the two groups. RESULTS: Approximately 35% of the patients were poor responders. Our analysis of the pretreatment clinical parameters revealed that number of anti-diabetic agents and use of sulfonylurea were significantly associated with poor response to liraglutide (P=0.02 and P=0.03, respectively) in a multivariate analysis. We were not able to find any candidate related to clinical response to exenatide. CONCLUSION: Our study showed that the therapeutic effects of GLP-1 analogues on T2DM patients were heterogeneous. T2DM patients who require multiple anti-diabetic agents, especially sulfonylurea, do not benefit from liraglutide treatment.

Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non-obese diabetic mice.

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of ß cells and perforin deficiency effectively reduces diabetes in non-obese diabetic (NOD) mice, it can be deduced that ß cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non-obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB(-/-) mice developed diabetes spontaneously with kinetics similar to those of wild-type NOD (wt-NOD) mice. Adoptive transfer study with regulatory T cell (Treg )-depleted splenocytes (SPCs) into NOD-SCID mice or in-vivo Treg depletion by anti-CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB(-/-) mice and wt-NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre-diabetic NOD.GzmB(-/-) mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide-promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4(+) , CD8(+) and CD4(+) CD25(+) T cells in SPCs from NOD.GzmB(-/-) mice than those from wt-NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for ß cell destruction in NOD mice.

Double deficiency in IL-17 and IFN-γ signalling significantly suppresses the development of diabetes in the NOD mouse.

AIMS/HYPOTHESIS: T helper type (Th) 17 cells have been shown to play important roles in mouse models of several autoimmune diseases that have been classified as Th1 diseases. In the NOD mouse, the relevance of Th1 and Th17 is controversial, because single-cytokine-deficient NOD mice develop diabetes similarly to wild-type NOD mice. METHODS: We studied the impact of IL-17/IFN-γ receptor double deficiency in NOD mice on the development of insulitis/diabetes compared with IL-17 single-deficient mice and wild-type mice by monitoring diabetes-related phenotypes. The lymphocyte phenotypes were determined by flow cytometric analysis. RESULTS: IL-17 single-deficient NOD mice showed delayed onset of diabetes and reduced severity of insulitis, but the cumulative incidence of longstanding diabetes in the IL-17-deficient mice was similar to that in wild-type mice. The IL-17/IFN-γ receptor double-deficient NOD mice showed an apparent decline in longstanding diabetes onset, but not in insulitis compared with that in the IL-17 single-deficient mice. We also found that double-deficient NOD mice had a severe lymphopenic phenotype and preferential increase in regulatory T cells among CD4(+) T cells compared with the IL-17 single-deficient mice and wild-type NOD mice. An adoptive transfer study with CD4(+)CD25(-) T cells from young non-diabetic IL-17 single-deficient NOD mice, but not those from older mice, showed significantly delayed disease onset in immune-deficient hosts compared with the corresponding wild-type mice. CONCLUSIONS/INTERPRETATION: These results indicate that IL-17/Th17 participates in the development of insulitis and that both IL-17 and IFN-γ signalling may synergistically contribute to the development of diabetes in NOD mice.

Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: changes with age and effects of cryptorchidism.

The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P < 0.05) from prepubertal age (4-6 mo) to pubertal age (6-12 mo), and then declined (P < 0.05) from pubertal age to post-pubertal age (1-5 y), reaching a plateau. Plasma testosterone concentrations increased (P < 0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P < 0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05-0.43 ng/ml) and testosterone (range: 0.10-0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL3 < 0.02 ng/ml; testosterone < 0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.

Isolation and characterization of a novel short peptide associated with Crohn's disease.

Phage display technology has been utilized to select target molecules against circulating antibodies. The aims of this study were to isolate a peptide that binds with serum from Crohn's disease (CD) patients and to examine its diagnostic and pathogenic significance. A phage display library was constructed using cDNA from Caco-2 cells. Affinity selection using this cDNA library and serum samples from patients with CD was then performed. Phage clones that specifically reacted with the CD sera were then selected using a phage enzyme-linked immunosorbent assay (ELISA). After the DNA sequences of the selected phages were determined and converted to amino acid sequences, the synthesized peptides were examined using an ELISA. The effect of the synthesized peptides on cytokine release from cultured blood mononuclear cells was investigated. An ELISA analysis for TCP-353 demonstrated that while 61·7% of the samples from CD patients were seroreactive, seroreactivity was less common among patients with ulcerative colitis (7·3%), acute colitis (0%) or colon cancer (11·4%) and among normal subjects (2·8%). The induction of interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α release, but not IL-10 release, in response to TCP-353 peptide was enhanced in CD mononuclear cells only. We isolated a novel peptide that specifically binds to CD sera and stimulates the proinflammatory responses of CD mononuclear cells. TCP-353 may have diagnostic, pathogenic and therapeutic significance with regard to the treatment of CD.

CHOP deletion does not impact the development of diabetes but suppresses the early production of insulin autoantibody in the NOD mouse.

C/EBP homologous protein (CHOP) has been proposed as a key transcription factor for endoplasmic reticulum (ER) stress-mediated ß-cell death induced by inflammatory cytokines in vitro. However, the contribution of CHOP induction to the pathogenesis of type 1 diabetes is not yet clear. To evaluate the relevance of CHOP in the pathogenesis of type 1 diabetes in vivo, we generated CHOP-deficient non-obese diabetic (NOD.Chop (-/-)) mice. CHOP deficiency did not affect the development of insulitis and diabetes and apoptosis in ß-cells. Interestingly, NOD.Chop (-/-) mice exhibited a delayed appearance of insulin autoantibodies compared to wild-type (wt) mice. Adoptive transfer with the diabetogenic, whole or CD8(+)-depleted splenocytes induced ß-cell apoptosis and the rapid onset of diabetes in the irradiated NOD.Chop (-/-) recipients with similar kinetics as in wt mice. Expression of ER stress-associated genes was not significantly up-regulated in the islets from NOD.Chop (-/-) compared to those from wt mice or NOD-scid mice. These findings suggest that CHOP expression is independent of the development of insulitis and diabetes but might affect the early production of insulin autoantibodies in the NOD mouse.

Thoracic mass in a cynomolgus macaque (Macaca fascicularis).

A male cynomolgus macaque at the age of 3 years and 11 months suffered sudden cardiac arrest during a surgical operation. This animal had been clinically asymptomatic for 6 months from the acclimatization period to death. At necropsy, a white mass approximately 5 cm in diameter was found at the base of the heart. Histopathologically, the mass consisted of a granuloma with a number of multinucleated giant cells and multiple necrotic foci. Fungal hyphae characterized by parallel cell walls, distinct septa, and branching were observed in the lesion. The granuloma extended into the thoracic lymph nodes and the subepicardium of the left atrium, compressed the bronchioli, and was separated from the pulmonary parenchyma by a thick fibrous layer. The hyphal morphology and results of polymerase chain reaction assays demonstrated that the pathogen was Aspergillus sp.

Intermittent administration of a sustained-release prostacyclin analog ONO-1301 ameliorates renal alterations in a rat type 1 diabetes model.

Diabetic nephropathy is the most common pathological disorder predisposing end-stage renal disease. ONO-1301 is a novel sustained-release prostacyclin analog possessing thromboxane (TX) synthase inhibitory activity. Here, we aimed to investigate the therapeutic efficacies of ONO-1301 in a rat type 1 diabetic nephropathy model. Streptozotocin (STZ)-induced diabetic rats received injections of slow-release form of ONO-1301 (SR-ONO) every 3 weeks. Animals were sacrificed at Week 14. SR-ONO significantly suppressed albuminuria, glomerular hypertrophy, mesangial matrix accumulation, glomerular accumulation of monocyte/macrophage, increase in glomerular levels of pro-fibrotic factor transforming growth factor (TGF)-beta1 and the number of glomerular alpha-smooth muscle actin (SMA)(+) cells in diabetic animals. The glomerular levels of hepatocyte growth factor (HGF) were significantly increased in SR-ONO-treated diabetic animals. Taken together, these results suggest the potential therapeutic efficacy of intermittent administration of SR-ONO in treating diabetic nephropathy potentially via inducing HGF, thus counteracting the pro-fibrotic effects of TGF-beta1.

Clinical trial: comparison of alendronate and alfacalcidol in glucocorticoid-associated osteoporosis in patients with ulcerative colitis.

BACKGROUND: Bone loss is often observed in patients with ulcerative colitis, particularly if they require glucocorticoids. AIM: To determine whether the bisphosphonate, alendronate, is safe and effective in preserving bone mass compared to the active vitamin D3, alfacalcidol, in ulcerative colitis patients receiving glucocorticoids. METHODS: Thirty-nine patients with ulcerative colitis and treated with glucocorticoids were randomized to receive alendronate (5 mg/day) or alfacalcidol (1 microg/day) daily for 12 months. Loss of bone mass was evaluated by bone mineral density, bone resorption by urinary N-telopeptide for type I collagen, and bone formation by serum bone alkaline phosphatase. RESULTS: Alendronate, but not alfacalcidol, significantly increased bone mineral density in the lumbar spine. Alendronate decreased serum bone alkaline phosphatase levels, but alfacalcidol did not. Urinary N-telopeptide for type I collagen levels decreased in both groups, but were significantly lower in the alendronate group. There were no significant differences in the adverse events in the two groups. CONCLUSION: Our study indicates that alendronate is a safe, well-tolerated and more effective therapy than alfacalcidol for preventing glucocorticoid-associated bone loss in patients with ulcerative colitis.

Association between anti-ZnT8 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes.

AIMS/HYPOTHESIS: We analysed the association between humoral autoreactivity to zinc transporter-8 (ZnT8) and the SLC30A8 rs13266634 polymorphism (Arg325Trp), which is located at the most distal loop in the ZnT8 protein. METHODS: Autoantibodies to ZnT8 were determined by RIA in 270 patients with type 1 diabetes using ZnT8 carboxy-terminal constructs (amino acids 268-369) carrying 325Trp(CW) and 325Arg(CR) and a hybrid construct (CW-CR). Forty-four ZnT8 autoantibody-positive sera with genomic DNA were used to examine the association between reactivity to ZnT8 constructs and the rs13266634 genotype. RESULTS: Seventy-five patients reacted to the CW-CR hybrid construct, whereas 37 and 36 patients reacted to the CW and CR constructs, respectively. All sera positive for either CW or CR autoantibodies were positive for CW-CR autoantibodies. Among 19 patients with a 325Arg(CC) genotype, 5% had CW-specific autoantibodies, 42% had CR-specific autoantibodies and 32% had dual reactivity. Conversely, 73% of 15 patients with the 325Trp(TT) genotype had CW-specific autoantibodies, no patients had CR-specific autoantibodies and 13% had dual reactivity. Nine of the ten patients (90%) with the CT genotype reacted with either CR or CW constructs. The titre of CR autoantibodies in patients carrying the C allele was significantly higher than that in TT homozygotes (p < 0.0001). In contrast, the titre of CW autoantibodies in patients carrying a T allele was significantly higher than that in CC homozygotes (p < 0.005). No evidence of an association between rs13266634 and type 1 diabetes was observed. CONCLUSIONS/INTERPRETATION: These results indicate that variant residue at amino acid 325 is a key determinant of humoral autoreactivity to ZnT8 and that the SLC30A8 genotype is an important determinant of autoantibody specificity.

Development of granulocyte and monocyte adsorptive apheresis in the rat dextran sodium sulfate-induced colitis model.

Granulocytapheresis (GCAP) selectively removes large numbers of granulocytes and monocytes from peripheral blood by adsorptive apheresis, and in patients with ulcerative colitis GCAP has been associated with significant efficacy. However, the mechanism(s) of efficacy of this strategy is poorly understood. This rat model of dextran sodium sulfate (DSS)-induced colitis was to investigate the effect of GCAP on tumor necrosis factor (TNF)-alpha release by peripheral leukocytes. By using mini columns, an experimental GCAP setting was developed and applied to the DSS-induced colitis model. The production of TNF-alpha by lipopolysaccharide-activated leukocytes in whole blood was measured by enzyme-linked immunosorbent assay. In rats that received GCAP with columns containing leukocytapheresis carriers, TNF-alpha release by leukocytes was significantly (p < 0.05) suppressed, while no change in TNF-alpha production was seen in rats that received GCAP with sham columns. This first experimental setting in the rat colitis model suggests that GCAP is feasible in animals and should shed light on the mechanism(s) of GCAP in clinical settings. Given that TNF-alpha is a major inflammatory cytokine, down-modulation of TNF-alpha might represent one mechanism of antiinflammatory effects of GCAP.

A form of circulating interleukin-6 receptor component soluble gp130 as a potential interleukin-6 inhibitor in inflammatory bowel disease.

The presence and the role of soluble gp130, the soluble form of a component of the interleukin (IL)-6 receptor complex, were investigated in inflammatory bowel disease. The serum concentrations of soluble gp130 were increased in ulcerative colitis (active disease, median, 93.5 ng/ml; interquartile range, 26-125 ng/ml; inactive disease, 81 ng/ml, 24.8-137.3 ng/ml) and to a lesser extent in Crohn's disease (active disease, 66 ng/ml, 44.4-87.6 ng/ml; inactive disease, 63 ng/ml, 43.5-82.5 ng/ml) compared to normal controls (43 ng/ml, 27-59 ng/ml). Paired analysis of serum samples showed a decrease of IL-6 and soluble IL-6 receptor concentrations in both diseases and an increase of soluble gp130 concentrations, especially in ulcerative colitis, just after the resolution of disease exacerbation. Size fractionation of the serum revealed that a part of the IL-6 co-eluted with soluble gp130 and soluble IL-6 receptor. The IL-6-induced proliferation of murine B9 hybridoma was enhanced by recombinant soluble IL-6 receptor, whereas the proliferation was inhibited by recombinant soluble gp130. These results indicate that soluble gp130 may function as a natural inhibitor of the IL-6 actions in inflammatory bowel disease.

Requirement of water-flow for sustainable growth of Pocilloporid corals during high temperature periods.

Reef-building corals are threatened worldwide by mass-scale coral bleaching episodes that are pronounced in high sea surface temperature (SST) conditions. Although water-flow has been suggested to be a mitigating factor for bleaching, long-term effects of flow-mediated bleaching suppression are as yet not fully understood. In order to investigate flow effects, we monitored the corals Pocillopora damicornis and Stylophora pistillata grown for 20 months in experimental outdoor flumes with the flow rates of 20 cms(-1) (flow) and <3 cms(-1) (still). Although bleaching was observed under high SST conditions, both species showed a shorter period or entirely no visible bleaching under the flow conditions. Better colony growth was found in the flow conditions whereas significant growth suppression and higher mortality were observed in still conditions. We conclude that water-flow is an essential environmental factor for the corals P. damicornis and S. pistillata, especially under high SST conditions.

Adenovirus-mediated gene delivery of interleukin-10, but not transforming growth factor beta, ameliorates the induction of Graves' hyperthyroidism in BALB/c mice.

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) are well known anti-inflammatory cytokines. We have studied the effect of adenovirus-mediated IL-10 and TGF-beta gene delivery on the induction of Graves' hyperthyroidism in our mouse model that involves repeated injections of adenovirus expressing the thyrotropin receptor A subunit (AdTSHR). We first constructed adenoviruses encoding the two cytokines (AdIL10 and AdTGF(beta)) and confirmed expression by in vitro infection of COS cells. Susceptible BALB/c mice were injected twice with AdTSHR alone or together with AdIL10 or AdTGF(beta), and bled two weeks after the second immunization. Significantly elevated serum thyroxine levels were seen in 26% of mice immunized with AdTSHR and AdIL10 versus 61% with AdTSHR alone. Levels of thyroid stimulating antibody, but not nonstimulating antibody, were also decreased, and TSHR-specific splenocyte secretion of interferon-gamma in recall assays was impaired in mice treated with AdIL10. In contrast, AdTGF(beta) had little effect on hyperthyroidism. Overall, our findings demonstrate that gene delivery of IL-10, but not TGF-beta, suppresses the induction of Graves' hyperthyroidism in a mouse model. However, the effect of IL-10 is less powerful than we observed previously with T helper type 2-inducers including adenovirus expressing IL-4, Shistosoma mansoni infection or alpha-galactosylceramide.