Differential effects of FK506 and methotrexate on inflammatory cytokine levels in rat adjuvant-induced arthritis

OBJECTIVE: To investigate the effects of prophylactic and therapeutic treatments with FK506 (tacrolimus), an immunosuppressive drug that specifically inhibits T cell activation, and methotrexate (MTX) on inflammatory cytokines, tumor necrosis factor (TNF)-a, interleukin (IL)-1beta, and IL-6 levels in rat adjuvant-induced arthritis (AIA). METHODS: AIA was induced in female Lewis rats. Arthritis was assessed by hindpaw swelling. TNF-a, IL-1beta, and IL-6 levels in paw extracts were determined by ELISA. To assess the effects on cytokine levels, rats were treated prophylactically with FK506 (3 mg/kg) or MTX (0.1 mg/kg) from day 1 to day 17, and therapeutically with FK506 (5 mg/kg) or MTX (1 mg/kg) from day 15 to day 17 (3-day treatment) or day 15 to 20 (6-day treatment) by oral administration. RESULTS: TNF-a, IL-1beta, and IL-6 levels in paw tissue were found to significantly increase between day 15 and day 21 after adjuvant injection, when the arthritis was in a developed stage. Prophylactic treatment with FK506 and MTX suppressed arthritis and reduced the levels of those inflammatory cytokines. FK506 caused a marked reduction of TNF-a and IL-1beta levels in paw tissue even in short-term (3-day) therapeutic treatment. It reduced all levels of TNF-a, IL-1beta, and IL-6 in paws in 6-day therapeutic treatment. In contrast, therapeutic treatment with MTX affected neither TNF-a or IL-6 levels in paws. MTX reduced IL-1beta levels only in the 6-day treatment. CONCLUSION: FK506 is more effective than MTX in reducing elevated levels of inflammatory cytokines TNF-a, IL-1beta, and IL-6 in established stages of AIA. Our findings suggest that inhibition of T cell activation results in a rapid reduction of inflammatory cytokine levels even after the arthritis is established in AIA.     

Detection and monitoring of methotrexate-associated lung injury using serum markers KL-6 and SP-D in rheumatoid arthritis

The applicability of monitoring concentrations of serum KL-6 and serum surfactant protein-D (SP-D) in the detection of methotrexate-associated lung injury (MTX pneumonitis) in patients with rheumatoid arthritis (RA) was investigated. The concentrations of these markers, sequentially measured in two patients with RA complicated with MTX pneumonitis, were increased in accordance with the severity of MTX pneumonitis. Conversely, the concentrations of these markers were decreased with the improvement of MTX pneumonitis, suggesting that the monitoring of these markers could be applicable not only for detecting the onset of MTX pneumonitis, but also for detecting the therapeutic response of MTX pneumonitis.     

Effect of nicorandil on coronary spasm

In patients in whom dynamic coronary obstruction plays a predominant role in the pathogenesis of angina pectoris, dilatation of large coronary arteries and relief of coronary spasm account for the main mechanism of action of antianginal drugs. In this study, the acute vasodilating effects of nicorandil, a newly developed antianginal drug, were assessed in 10 patients who had spontaneous and ergonovine-evoked coronary spasms. The prompt, complete relief of both spontaneous and evoked coronary spasms was obtained in all of 10 patients with nicorandil. The coronary spasmolytic effect of nicorandil in the present series is thus considered to be beneficial to the treatment of coronary spasm.     

Unitary step of gliding machinery in Mycoplasma mobile

Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0–4.5 μm⋅s⁻¹ with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.The fastest of the Mycoplasma species is Mycoplasma mobile (M. mobile); they glide with a speed of 2.0–4.5 μm⋅s⁻¹ (1, 2). Under an optimal-growth condition, cultivated single M. mobile cells are flask-shaped (Fig. 1A) and glide smoothly across a substrate covered with surface-immobilized sialylated oligosaccharides (3) in the direction of protrusion at a constant speed (Movie S1). Genomic sequencing and analysis have revealed that the mechanism must differ from other forms of motor protein systems and bacterial motility, because M. mobile lacks genes encoding conventional motor proteins in eukaryotes, such as myosin, kinesin, and dynein, in addition to lacking other motility structures in bacteria, such as flagella and pili (4). So far, three proteins have been identified as a part of the gliding machinery (Fig. 1B, Bottom): Gli123 (5), Gli521 (6), and Gli349 (7). The machinery units localize around the cell neck, and their number has been estimated to be ∼450 (2, 5, 8). Gli349 extends out from the cell membrane and shows a rod structure, ∼100 nm in total, with two flexible hinges when isolated (9). Notably, the machinery is driven by hydrolysis of ATP to ADP and inorganic phosphate, caused by an unknown ATPase (10). Because of the large size and characteristic structure of Gli349, and a series of studies with mutants and inhibitory antibodies (2, 11), it has been hypothesized that Gli349 works as a “leg” by binding to and releasing from a substrate covered with randomly arranged sialylated oligosaccharides (2) consuming the chemical energy of ATP. In addition, the pivoting movement of an elongated cell suggests that there are units working not simultaneously but rather independently to propel the cell forward (12). To test this hypothesis and identify conformational changes of a key part of the gliding machinery, we here designed an assay to detect the movement of M. mobile by high precision colocalization microscopy. In the presence of an excess number of binding targets in the solution, which decreased the number of active legs, stepwise displacement was shown for the first time, to our knowledge, to occur in gliding bacteria.Open in a separate windowFig. 1.Nanometer-scale tracking of Mycoplasma gliding. (A) A dark-field image of M. mobile. The image was captured with center-stop optics to maintain the high numerical aperture of the objective, which enabled a high spatial resolution (35). (Scale bar: 1 μm.) (B, Upper) Illustration of the fluorescent ghost. The gliding machinery was distributed around the neck portion, but only the active machinery bound to the glass is shown for simplicity. (Bottom) A construction model of the gliding machinery comprising three proteins: Gli123, Gli521, and Gli349. See the review by Miyata (2) for more detail. (C) A fluorescent image of the labeled ghost was acquired with a time resolution of 2 ms. (Scale bar: 1 μm; pixel size: 240 nm.) (D) The intensity profile of C. The XY area is 5 × 5 μm. (E) Gaussian fitting to D. Nanometer-scale tracking is achieved by positioning the peak of the 2D Gaussian function fitting to the intensity profile of the ghost. (F, Left) The speed of gliding ghosts at different [ATP]s in the solution (n = 129). The cyan curve shows a fit with Michaelis–Menten kinetics; Vmaxspeed and K_m are 2.6 µm⋅s⁻¹ and 61 µM, respectively. The dotted cyan curve shows a fit with the kinetics including the Hill coefficient; Vmaxspeed, [ATP₅₀] and n are 2.2 µm⋅s⁻¹, 43 µM, and 2.4, respectively. (Right) The speed of living cells with no ATP in the solution (2.1 ± 0.1 µm⋅s⁻¹; n = 22). (G) Effect of SL on the gliding velocity of the ghost at saturated [ATP]s, 0.3–1.0 mM (n = 50).     

Exposure to intermittent hypoxia inhibits allergic airway inflammation in a murine model of asthma

Sleep and Breathing - Obesity increases the severity of asthma, and patients with severe asthma are often complicated with obstructive sleep apnea syndrome (OSAS), a concomitant disease of obesity....     

Skin Autofluorescence Predicts Cardiovascular Mortality in Patients on Chronic Hemodialysis

Tissue accumulation of advanced glycation end products (AGE) is thought to contribute to the progression of cardiovascular disease (CVD). Skin autofluorescence, a non‐invasive measure of AGE accumulation using autofluorescence of the skin under ultraviolet light, has been reported to be an independent predictor of mortality associated with CVD in Caucasian patients on chronic hemodialysis. The aim of this study was to assess the predictive value of skin autofluorescence on all‐cause and cardiovascular mortality in non‐Caucasian (Japanese) patients on chronic hemodialysis. Baseline skin autofluorescence was measured with an autofluorescence reader in 128 non‐Caucasian (Japanese) patients on chronic hemodialysis. All‐cause and cardiovascular mortality was monitored prospectively during a period of 6 years. During the follow‐up period, 42 of the 128 patients died; 19 of those patients died of CVD. Skin autofluorescence did not have a significant effect on all‐cause mortality. However, age, carotid artery intima‐media thickness (IMT), serum albumin, high‐sensitivity C‐reactive protein (hsCRP), skin autofluorescence and pre‐existing CVD were significantly correlated with cardiovascular mortality. Multivariate Cox regression analysis showed skin autofluorescence (adjusted hazard ratio [HR] 3.97; 95% confidence interval [CI]1.67–9.43), serum albumin (adjusted HR 0.05; 95% CI 0.01–0.32), and hsCRP (adjusted HR 1.55; 95% CI 1.18–2.05) to be independent predictors of cardiovascular mortality. The present study suggests that skin autofluorescence is an independent predictor of cardiovascular mortality in non‐Caucasian (Japanese) patients on chronic hemodialysis.