Changes in serum complements and their regulators in generalized myasthenia gravis.

OBJECTIVE: To investigate changes in serum complements and their regulators in the pathogenesis of myasthenia gravis (MG). METHODS: Forty-four patients with acetylcholine receptor antibody-positive MG, as well as 20 patients with non-inflammatory neurological disorders were enrolled. Serum complements (C3, C4 and soluble C5b-9) and complement regulators (vitronectin, clusterin and properdin) were extensively analysed by enzyme-linked immunosorbent assay and their associations with clinical profiles of MG were examined. RESULTS: Serum C3, C4 and clusterin levels were not significantly different between patients with MG and controls. The patients with MG had higher soluble C5b-9 (P = 0.09) and vitronectin (P = 0.001) levels than the controls; moreover, vitronectin levels decreased after treatment (P = 0.09). Serum properdin (P = 0.03) levels were lower in the patients with MG than in the controls, and negatively correlated with the MG Activities of Daily Living score (rs = -0.26, P = 0.09) and with the presence of bulbar palsy (P = 0.04). CONCLUSION: Our results show that activation of complements and an altered complement network could contribute to the inflammatory pathogenesis of MG.

Serum level of soluble urokinase plasminogen activator receptor (suPAR) as a disease severity marker of myasthenia gravis: a pilot study.

Myasthenia gravis (MG) is an autoantibody-mediated inflammatory disease of the neuromuscular junction. Biomarkers indicating disease activity in MG are warranted. Recently, the soluble urokinase plasminogen activator receptor (suPAR) has been reported to be associated with inflammation, tissue damage, disease activity and prognosis in various diseases, including autoimmune diseases. In this study, serum suPAR levels were measured in 40 patients with anti-acetylcholine receptor antibody-positive MG and 30 controls, and their correlations with clinical variables and severity scale scores were investigated. We identified that serum suPAR levels significantly correlated with MG activities of daily living scale (Spearman's ρ = 0·45; P = 0·004) and MG Foundation of America classification (Spearman's ρ = 0·37; P = 0·02) at serum sampling, but not with anti-acetylcholine receptor antibody titers. In conclusion, serum suPAR levels can be a candidate for a novel biomarker of disease activity in anti-acetylcholine receptor antibody-positive MG.

Frequency and features of myasthenia gravis developing after thymectomy.

BACKGROUND AND PURPOSE: Thymectomy is an effective treatment for myasthenia gravis (MG) with anti-acetylcholine receptor (AChR) antibodies. We rarely encounter patients who develop MG after surgery for thymic tumors. This study aimed to investigate the characteristics and frequency of post-thymectomy onset (PostTx) MG. METHODS: We reviewed the clinical information of thymoma-associated MG in 158 patients. Of these, 18 (11%) patients with PostTx MG were identified. RESULTS: The presence of anti-AChR antibodies (82%) and electrophysiological abnormalities (50%) was confirmed before thymectomy in patients with PostTx MG. The clinical characteristics of PostTx MG were similar to those of pre-thymectomy onset (PreTx) MG. In PostTx MG, the duration between thymectomy and MG onset were distributed as < 6 months (early-onset PostTx MG) and ≥ 6 months (late-onset PostTx MG). Notably, some patients with late-onset PostTx MG were associated with thymoma relapse. CONCLUSION: Our results suggest that approximately 11% of patients with thymoma-associated MG were PostTx MG and pre-surgical assessment of anti-AChR antibody titer or electrophysiological testing may predict PostTx MG development. However, no difference in clinical manifestation and prognosis was observed between PreTx MG and PostTx MG.

Safety of tapering tacrolimus dose in patients with well-controlled anti-acetylcholine receptor antibody-positive myasthenia gravis.

BACKGROUND AND PURPOSE: Tapering immunosuppressants is desirable in patients with well-controlled myasthenia gravis (MG). However, the association between tapering of calcineurin inhibitor dosage and reduction-associated exacerbation is not known. The aim of this study was to clarify the frequency of reduction-associated exacerbation when tacrolimus is tapered in stable patients with anti-acetylcholine receptor antibody-positive MG, and to determine the factors that predict exacerbations. METHODS: We retrospectively analyzed 115 patients in whom tacrolimus dosage was tapered. The reduction-associated exacerbation was defined as the appearance or worsening of one or more MG symptoms <3 months after the reduction. RESULTS: Tacrolimus dosage was successfully tapered in 110 patients (96%) without any exacerbation. Five patients (4%) experienced an exacerbation, but symptoms were reversed in all patients when the tacrolimus dose was increased to the previous maintenance level. No patient developed an MG crisis. The age at onset was significantly earlier (30 vs. 56 years, P = 0.025) and the reduction in dosage was significantly larger (2.0 vs. 1.0 mg/day, P = 0.002) in patients with reduction-associated exacerbation than in those without exacerbation. The cut-off values determined in a receiver-operating characteristic curve analysis were 52 years (sensitivity, 57%; specificity, 100%) for the age at onset and 1.5 mg (sensitivity, 80%; specificity, 100%) for the dose reduction. CONCLUSION: Tapering of tacrolimus was possible in most patients with well-controlled anti-acetylcholine receptor antibody-positive MG. Early age at onset and a large reduction from maintenance dosage were associated with exacerbation. Reductions ≤1.5 mg/day from the maintenance dosage should be considered for patients with late-onset disease.

Adequate tacrolimus concentration for myasthenia gravis treatment.

BACKGROUND AND PURPOSE: A single, oral dose of 3 mg/day tacrolimus, approved for myasthenia gravis (MG) treatment in Japan, was shown to reduce steroid dose and anti-acetylcholine receptor (AChR) antibody titers as well as to improve MG symptoms. However, no studies have investigated the association between tacrolimus concentration and its clinical efficacy in MG. In this study, we aimed to determine the optimal tacrolimus concentration for MG treatment. METHODS: The trough tacrolimus concentration in 51 patients with MG (positive for anti-AChR antibody, n = 48; negative for anti-AChR and anti-muscle-specific tyrosine kinase antibodies, n = 3) who received 3 mg/day tacrolimus for more than 1 year was measured using a chemiluminescent enzyme immunoassay. The clinical characteristics of patients with MG as well as the dose of prednisolone used before and after tacrolimus treatment were evaluated retrospectively. RESULTS: The median trough tacrolimus concentration was 5.4 (range, 2.9-7.6) ng/mL, which was correlated with 'minimal manifestation or better status' (P = 0.0190, r = 0.3273) and the reduction in anti-AChR antibody 1 year after tacrolimus initiation (P = 0.0170, r = 0.3465). When the cut-off value for tacrolimus was defined as 4.8 ng/mL using a receiver operating characteristic curve, patients with adequate tacrolimus concentration (≥4.8 ng/mL) showed more reduction in anti-AChR antibody titers and more improvement in MG-related activities in daily life scores. More patients with adequate tacrolimus concentration achieved 'minimal manifestation or better status' compared with those with low tacrolimus concentration. CONCLUSIONS: An adequate tacrolimus concentration is required for better MG prognosis.

Interaction between gastrocnemius muscle weakness and moderate exercise deteriorates joint integrity in rat knee.

The aim of this study was to determine the effect on the knee joint of the interaction between ankle muscle weakness and moderate exercise. Gastrocnemius muscle weakness was induced by intramuscular injection of botulinum toxin type A (BTX) in rats. Low-speed treadmill running (12 m/min for 60 min) was applied for 6 weeks in rats with and without BTX. Untreated animals were used as controls. After BTX injection, the gastrocnemius muscle weakness was confirmed by 3-D motion analysis in kinematic features of the hindlimb during locomotion as an increased maximal dorsiflexion angle during the stance phase. Serum biomarker analysis by enzyme-linked immunosorbent assay revealed that low-speed running decreased the catabolic effect on type II collagen. However, the inhibition of catabolism induced by running exercise was significantly counteracted by BTX injection. In addition, thinning of the cartilage layer and a reduction in the chondrocyte density was also found in the tibial plateau of the knee in the BTX-injected rats after running for 6 weeks. These data suggest that moderate exercise have a positive effect on joint homeostasis. However, ankle muscle weakness may alter the mechanical environment of the knee and impair the integrity of joint cartilage with moderate exercise.

Serum cytokine and chemokine profiles in patients with myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune-mediated inflammatory disease of the neuromuscular junction. Previous studies of animal MG models have suggested important roles of cytokines in MG pathogenesis, but adequate studies on cytokines in human MG are lacking. Using a multiplex suspension array system, we measured the serum levels of 27 cytokines/chemokines in 47 anti-acetylcholine receptor antibody-positive patients with MG and 20 normal controls (NC) to investigate the contribution of cytokines/chemokines toward MG pathogenesis. Correlations between clinical parameters and cytokine/chemokine levels in patients with MG were also examined. The serum levels of interleukin (IL)-15 (mean ± standard deviation: 6·85 ± 6·97 pg/ml) and vascular endothelial growth factor (VEGF) (96·21 ± 71·60 pg/ml) significantly increased, whereas IL-4 levels (3·57 ± 0·86 pg/ml) decreased in patients with MG compared with NC (IL-15: 4·42 ± 1·55 pg/ml; VEGF: 63·51 ± 32·95 pg/ml; IL-4: 4·15 ± 0·81 pg/ml, P < 0·05). In addition, eight cytokines (IL-4, IL-8, IL-15, eotaxin, macrophage inflammatory protein-1α, macrophage inflammatory protein-1ß, VEGF and IL-1b) were significantly changed among MG patients with thymoma, MG patients without thymoma and NC (P < 0·05). Some cytokines, such as IL-4, IL-15, and VEGF, may play roles in the pathogenesis of MG.

Data-driven cascade control system: Response estimation and controller design.

In this study, we propose a data-driven design method for a cascade control system with inner and outer control loops. First, the input-output response of a controlled plant, which varies with the controller parameters of a fixed-structure inner-outer control law, is estimated directly from open-loop input-output data. Next, based on the estimated response, the controller parameters are optimized to minimize the difference between a reference model with a controlled closed-loop system. In the proposed method, the response of a fictitious reference input, which varies with the controller parameters, is estimated, and then the closed-loop response is estimated. Therefore, a closed-loop input-output data is not required, and the controller parameters are determined directly from an open-loop input-output data. Furthermore, the time constant of a reference model is also optimized so as to reduce the control error. The proposed method is compared with both conventional single-loop and cascade data-driven methods by means of numerical examples.

Data-driven control for multi-rate multi-input/single-output systems.

For the next generation of manufacturing, represented by Industrie 4.0, a multi-input controller is designed directly from controlled data, without using the mathematical plant model, where the ratio between the D/A conversion of multiple inputs and the A/D conversion of a single output is non-uniquely. With the proposed method, the fixed-structured controller is optimally designed by solving a model reference problem using one-shot data. Furthermore, to eliminate inter-sample ripples emerged by input oscillation, the deviation of the control inputs is also evaluated using the proposed method. As a result, a non-ripple data-driven controller is achieved. Numerical examples show that the proposed multi-rate data-driven method is superior than the conventional single-rate method.

Very long baseline interferometric observations made with an orbiting radio telescope.

An orbiting spacecraft and ground observatories have been used to obtain interferometric observations of cosmic radio sources. The Tracking and Data Relay Satellite System (TDRSS) was used as the orbiting observatory in conjunction with two 64- meter radio telescopes at ground observatories, one in Australia and one in Japan. The quasars 1730-130 (NRAO 530), 1510-089, and 1741-038 were observed at a frequency of 2.3 gigahertz, and a maximum projected baseline of 1.4 earth diameters was achieved. All quasar observations for which valid data were acquired resulted in detected fringes. Many of the techniques proposed for a dedicated very long baseline interferometry observatory in space were used successfully in this experiment.

[Extensive educational program for high performance medical technologists].

The education system for medical technologists has recently been revolutionized, their educational periods vary from 2 to 9 years, and some already have doctoral degrees. In such a new situation, our faculty thinks that the most important point for new medical technologists is the ability to have a broad view of the clinical fields, especially the view of patients. Special training in bed-side education and a stint in several divisions, such as the surgical operation room, rehabilitation. radiological examination room, pharmacy, central storage room of medical records, and medical informatics, and so on, of the hospital is a powerful tool to obtain a broad view of the various clinical fields and can be essential for developing high performance medical technologists. As nine years have passed since starting this education, we evaluated this practice through systematic personal communication. As a result, it was found to be extremely effective for many reasons such as having a continuous image of the patient when they examine the blood sample in the hospital laboratory, showing advanced laboratory performance, and having no mental barrier to visiting the wards and so on. The abilities of our alumni are praised highly by many large scale hospitals around the country and 50% of them are working in the clinical laboratory division of these hospitals. About 40% are working in the division of research and development in various companies. We express sincere thanks to the director and all cooperative individuals for this course in the Osaka University Hospital.

Allelotype analysis in osteosarcomas: frequent allele loss on 3q, 13q, 17p, and 18q.

We have investigated the involvement of tumor suppressor genes in the genesis of osteosarcoma by analyzing allele losses at polymorphic loci in tumor tissues. Genotypes of DNA from primary osteosarcoma tissue and corresponding normal cells from 37 patients were analyzed at 58 polymorphic loci representing each autosomal chromosome arm except 5p and 20q. Allele losses were found at polymorphic loci on 36 of 37 chromosome arms analyzed. In particular, four of them showed frequencies of allele loss higher than 60%: 3q (75%); 13q (68%); 17p (72%); and 18q (64%). This result suggests that, in addition to the RB (retinoblastoma) gene on 13q and the p53 gene on 17p, at least two more tumor suppressor genes located on 3q and 18q are frequently involved in the development of osteosarcoma. The extent of allele losses as defined by fractional allelic loss among 36 tumors was diverse, from 0 to 0.64. The median fractional allelic loss value of 0.32 was much higher than those previously reported in colorectal carcinoma and breast carcinoma. Although no definite association of fractional allelic loss value to clinical prognosis of each case was found in osteosarcoma, tumors with 17p loss were more prone to the early onset of lung metastasis than tumors without 17p loss, indicating that allele loss on chromosome 17p can be a useful measure of prognosis.

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts.

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.

Retardation in bone resorption after bone marrow ablation in klotho mutant mice.

The klotho gene mutant mice exhibit both osteopetrotic phenotype, including elongation of trabeculae in the epiphyses of long bones and vertebral bodies, and osteopenic phenotype, such as thin cortical bones in the diaphyses of these bones. These diverse features raise the question of whether the klotho gene defect results in alteration in bone resorption in vivo. Therefore, we examined the effect of the klotho gene defect on bone resorption by using bone marrow ablation model. At 1 week after bone marrow ablation, trabecular bones were formed in the ablated marrow cavity to levels higher than those in unablated bones in both klotho mutant and wild-type mice. At 2 weeks postsurgery, newly formed trabecular bones were resorbed in wild-type mice to resume normal bone marrow and trabecular bone volume fraction as reported previously. In contrast, the newly formed trabecular bones in the ablated marrow in klotho mutant mice remained at levels similar to those at 1 week. The defect in the bone resorption phase in klotho mutant mice is associated with site-specific reduction of the number and size of osteoclasts in klotho mutant mice. Moreover, the expression levels of osteoprotegerin messenger RNA in the ablated femora of klotho mutant mice were higher than those in wild-type mice. These results indicate that lack of klotho gene expression suppressed bone resorption that should normally take place 2 weeks after bone marrow ablation.

Role of angiotensin II in high fructose-induced left ventricular hypertrophy in rats.

Recent studies suggest the linkage of hypertension and insulin resistance. High fructose diet is known to induce hyperinsulinemia and hypertension in rats. In a previous study, however, high fructose (66%) diet failed to elevate blood pressure but increased left ventricular weight in Sprague-Dawley rats. In the present study, we investigated the precise mechanism of high fructose diet-induced changes in the cardiovascular system in rats. Intake of fructose-enriched diet for 2 weeks increased serum insulin and plasma angiotensin II levels. Urinary excretion of sodium and norepinephrine was not changed. Blood pressure measured directly through an indwelling catheter was not increased, but left ventricular weight and protein content were increased by high fructose diet. To further elucidate the role of the renin-angiotensin system, an angiotensin II type 1 receptor antagonist, TCV-116, was given orally at 1 mg/kg per day with either normal or high fructose diet. Concomitant administration of TCV-116 did not affect plasma glucose or serum insulin levels. Plasma angiotensin II was increased, but neither urinary sodium nor norepinephrine was changed by TCV-116. TCV-116 similarly decreased blood pressure in rats on normal and high fructose diets. Increase in left ventricular weight induced by high fructose diet was prevented by the concomitant administration of TCV-116. On the other hand, left ventricular weight in control rats was not changed by TCV-116. In conclusion, increased plasma angiotensin II may account for the left ventricular hypertrophy induced by high fructose diet, whereas hemodynamic change, sodium retention, and the sympathetic nervous system do not play an important role.

Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia?

Localization of the mRNAs for fractalkine, a CX3C chemokine, and for its receptor CX3CR1 was investigated in the rat brain. In situ hybridization study revealed that fractalkine mRNA was dominantly expressed in neuronal cells particularly in the olfactory bulb, cerebral cortex, hippocampus, caudate putamen and nucleus accumbens. In vitro study using enriched neuronal or glial culture supported the dominant expression of fractalkine mRNA in neurons. On the other hand, CX3CR1 mRNA was dominantly expressed in glial cells throughout the whole brain. The in vitro study suggested the cells expressing CX3CR1 mRNA are microglia, not astrocytes or neurons. Fractalkine appears to function as a signal molecule from neuron to microglia.

Serum levels of tumor necrosis factor-alpha in chronic inflammatory demyelinating polyneuropathy.

BACKGROUND: Activated macrophages and T lymphocytes may play a role in the pathogenesis of chronic inflammatory demyelinating polyneuropathy (CIDP). Both cell types secrete tumor necrosis factor-alpha (TNFalpha), which has toxic effects on myelin and endothelial cells. METHODS: The serum concentration of TNFalpha was measured by ELISA and compared with clinical and electrophysiological profiles in 20 patients with CIDP. RESULTS: An increased serum level of TNFalpha was detected in 5 (25%) patients and was associated with subacute progression, severe neurologic disabilities, and symmetric weakness involving proximal as well as distal muscles. TNFalpha levels increased during the active phase in this subgroup of patients. The levels of TNFalpha correlated with the severity of demyelinating conduction abnormalities in the intermediate as well as distal nerve segments, suggesting demyelination diffusely distributed along the nerves. CONCLUSION: Circulating TNFalpha increases during the active phase in a subgroup of CIDP patients and may play a role in the pathogenesis of demyelination and the breakdown of the blood-nerve barrier in CIDP.

Transcription factors and osteoblasts.

Transcription factors play a key role in determination of the fate of the cells in osteoblastic and chondrocytic lineage. A runt family member, Cbfa, is indispensable for osteoblastic differentiation. Sox 9 and scleraxis are involved in the phenotypic expression in chondrocytes and the cells of early stage connective tissues. These transcription factors will give us a clue to unravel interaction of these known and yet unknown transcription factors to fully understand the mechanisms of skeletal cells ' differentiation and regulation of their functions.

FK506-induced kidney tubular cell injury.

Some renal changes associated with cyclosporine, such as tubular vacuolization and glomerular thrombosis, have also been reported with FK506. Furthermore, FK506 therapy is associated with a decrease in glomerular filtration rate and renal plasma flow and an increase in renal vascular resistance. We studied the in vitro tubular cell sensitivity to FK506 in comparison with CsA, the ultrastructural changes induced by FK506 and CsA, and the effect of both drugs on tubular cell growth in vitro. We also investigated whether FK506 and CsA induced endothelin-1 (ET-1) secretion of cultured tubular cells and whether this stimulatory effect coincided with a change in the endothelin systemic synthesis. Exposure of tubular cells to high concentrations of FK506 or CsA (10, 50, 100 microM) induced a time- and dose-dependent cell injury in vitro. The damage induced by FK506 and CsA was characterized by a direct cytotoxic effect on tubular cells, as expressed by release of 3H thymidine from prelabeled cells, N-acetyl-beta-D-glucosaminidase release, and cell detachment. Ultrastructural changes (vacuolizations, swelling, and mitochondrial enlargement) and inhibition of the growth of cultured tubular cells were also observed at high concentrations of FK506 and CsA. Low concentrations of FK506 and CsA (1, 0.1, 0.01, 0.001 microM) were not cytotoxic and induced only a minimal inhibitory effect on the growth of tubular cells in vitro. We demonstrated that FK506 (1, 0.1, 0.01 microM) time-dependently stimulated the secretion of endothelin by cultured tubular cells. CsA 10, 1, 0.1, 0.01 also exerted an enhancing effect on ET-1 secretion in cultured tubular cells. We observed that the concentration of CsA that induced the most important enhancing effect was 10 or 100 times higher than that required for FK506 to observe the same effect. The concentrations of FK506 or CsA that induced ET-1 secretion were not cytolytic for tubular cells in vitro. FK506- or CsA-treated rats showed an increase in serum level of ET-1 in comparison with the control. Through the stimulatory effect on endothelin secretion by tubular cells, FK506 and CsA may induce a perturbation of renal hemodynamics. Concentrations of FK506 and CsA, higher than established serum levels but close to those reached in tissues, are cytotoxic for tubular cells and induced ultrastructural changes and a significant delayed regeneration.

Klotho-deficient mice are resistant to bone loss induced by unloading due to sciatic neurectomy.

Unloading induces bone loss as seen in experimental animals as well as in space flight or in bed-ridden conditions; however, the mechanisms involved in this phenomenon are not fully understood. Klotho mutant mice exhibit osteopetrosis in the metaphyseal regions indicating that the klotho gene product is involved in the regulation of bone metabolism. To examine whether the klotho gene product is involved in the unloading-induced bone loss, the response of the osteopetrotic cancellous bones in these mice was investigated. Sciatic nerve resection was conducted using klotho mutant (kl/kl) and control heterozygous mice (+/kl) and its effect on bone was examined by micro-computed tomography (microCT). As reported previously for wild-type mice (+/+), about 30% bone loss was induced in heterozygous mice (+/kl) by unloading due to neurectomy within 30 days of the surgery. By contrast, kl/kl mice were resistant against bone loss induced by unloading after neurectomy. Unloading due to neurectomy also induced a small but significant bone loss in the cortical bone of the mid-shaft of the femur in the heterozygous mice; no reduction in the cortical bone was observed in kl/kl mice. These results indicate that klotho mutant mice are resistant against bone loss induced by unloading due to neurectomy in both cortical and trabecular bone and indicate that klotho is one of the molecules involved in the loss of bone by unloading.