Real-time monitoring of mesangial cell-macrophage cross-talk using SEAP in vitro and ex vivo

BACKGROUND: Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. We established a novel system for continuous, real-time monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. METHODS: Rat mesangial cells were genetically engineered to produce secreted alkaline phosphatase (SEAP) under the control of the nuclear factor-kappaB (NF-kappaB) enhancer elements. The established sensor cells were exposed to macrophages or macrophage-derived factors, and the level of SEAP production was evaluated. RESULTS: In vitro, the established cells expressed and secreted SEAP when exposed to activated macrophages or to cytokines produced by macrophages. The kinetics of SEAP activity in culture media was closely correlated with the expression level of SEAP mRNA. The sensor cells also secreted SEAP in response to media conditioned by macrophage-accumulating, inflamed rat glomeruli. When the sensor cells were transferred adoptively into rat glomeruli subjected to acute anti-Thy 1 glomerulonephritis, the isolated glomeruli containing sensor cells secreted SEAP rapidly and progressively. CONCLUSION: These data suggested that the established system provides simple and useful tools for monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. This approach would be useful for investigation of molecular mechanisms involved in mesangial cell-macrophage interaction and also for screening of therapeutic agents that efficiently interfere with the link between infiltrating leukocytes and resident glomerular cells.     

Comparison between coefficient of R-R interval variation and gastric emptying in type 2 diabetes mellitus patients

BACKGROUND: Autonomic neuropathy is a common complication of diabetes mellitus (DM). METHOD: To clarify the relationship between cardiovascular autonomic function and gastric emptying rate, we investigated the gastric emptying and coefficient of R-R interval variation (CV(RR)) of 84 type 2 diabetic patients: 28 cases without peripheral neuropathy and 56 cases with peripheral neuropathy. All patients were subjected to a gastric emptying test according to the marker method (administration of a capsule containing 20 pieces of radiopaque marker during breakfast, followed by abdominal X-ray imaging 3 and 5 h later). Patients had their CV(RR) assessed at rest and during deep breathing. RESULTS: Gastric emptying scores were significantly correlated with CV(RR) during deep breathing and with the duration of DM, but neither age nor CV(RR) at rest in all patients. Gastric emptying scores and CV(RR) at rest and during deep breathing in patients with peripheral neuropathy were significantly deteriorated than those in patients without peripheral neuropathy. A significant correlation between gastric emptying and CV(RR) during deep breathing could be observed in the patients with peripheral neuropathy, but not in those without it. CONCLUSIONS: These findings showed that CV(RR) during deep breathing might be a good indicator of diabetic gastropathy and that peripheral neuropathy was closely related with cardiac and gastric autonomic neuropathy in the type 2 diabetic patients.     

Synergistic antithrombotic effect of a combination of NO donor and plasma kallikrein inhibitor

The aim of the present study was to investigate the effect of a NO donor (GSNO) and a plasma kallikrein inhibitor (PKSI-527) alone and in combination on global haemostatic status. A new in vitro test was employed which allows the measurement of both platelet function and spontaneous thrombolysis. Sixteen healthy young and 18 elderly volunteers were enrolled in this study. When GSNO (1 mM) or PKSI-527 (20 microM) was added to native human blood, platelet reactivity was significantly inhibited in both age groups. The combination of GSNO and PKSI-527 had additive inhibitory effect on platelets. Addition of either GSNO or PKSI-527 to blood samples did not significantly affect spontaneous thrombolysis, while added together, spontaneous thrombolysis was significantly enhanced. The thrombolysis enhancing effect was more prominent in elderly subjects. Our present findings suggest that the combination of NO donor and plasma kallikrein inhibitor may have clinical antithrombotic potential.     

Cognitive impairment after traumatic brain injury: a functional magnetic resonance imaging study using the Stroop task

The anterior cingulate cortex (ACC) plays a key role in cognition, motor function, and emotion processing. However, little is known about how traumatic brain injury (TBI) affects the ACC system. Our purpose was to compare, by functional magnetic resonance imaging (fMRI) studies, the patterns of cortical activation in patients with cognitive impairment after TBI and those of normal subjects. Cortical activation maps of 11 right-handed healthy control subjects and five TBI patients with cognitive impairment were recorded in response to a Stroop task during a block-designed fMRI experiment. Statistical parametric mapping (SPM99) was used for individual subjects and group analysis. In TBI patients and controls, cortical activation, found in similar regions of the frontal, occipital, and parietal lobes, resembled patterns of activation documented in previous neuroimaging studies of the Stroop task in healthy controls. However, the TBI patients showed a relative decrease in ACC activity compared with the controls. Cognitive impairment in TBI patients seems to be associated with alterations in functional cerebral activity, especially less activation of the ACC. These changes are probably the result of destruction of neural networks after diffuse axonal injury and may reflect cortical disinhibition attributable to disconnection or compensation for an inefficient cognitive process.     

Quantitative analysis of radioisotope cisternography in the diagnosis of intracranial hypotension

OBJECT: The authors attempted a quantitative analysis of conventional radioisotope cisternography for the purpose of more accurate diagnosis of intracranial hypotension. METHODS: Fifty-seven patients suspected of having intracranial hypotension underwent radioisotope cisternography. Whole-body images were obtained 2.5, 6, and 24 hours after intrathecal injection of 111In-diethylenetriamine pentaacetic acid. Radioactivity in the cerebrospinal fluid (CSF) space was counted during scanning, and radioisotope clearance was studied. Direct signs of radioisotope leakage into the spinal epidural space were found in 25 patients. Most leaks were located in the lumbosacral region. Analysis of the radioisotope clearance curve revealed two different patterns. In patients without a radiographically demonstrated radioisotope leak, absolutely exponential curves were observed (R2 > 0.99). The activity of the radioisotope decreased at a rate of e(-003) to e(-0.107) (mean +/- standard deviation, e(-0.056 +/- 0.018); 32 patients). Clearance in patients with an overt radioisotope leak was not a simple exponential curve; it could be divided into an early rapid phase and a late slow phase. The clearance rate during the first 6 hours was e(-0.219 +/- 0.127) (25 patients) and e(-0.076 +/- 0.021) thereafter. The authors speculated that the early escape of undiluted radioisotope solution through an aberrant CSF outlet, such as a traumatic spinal dural tear, was responsible for this phenomenon. CONCLUSIONS: The quantitative analysis featured in this study seems to be useful in the diagnosis of intracranial hypotension. A small CSF leak below the limit of radioisotope cisternography resolution might be detected using this technique.     

Optical mapping of neural responses and their gamma-aminobutyric acid-ergic inhibitory effects in the auditory brainstem of early postnatal mice

gamma-Aminobutyric acid (GABA)ergic neurons play important tropic and modulatory roles in the auditory pathway, especially in the early stage between postnatal Days 0 and 5. The effects of GABA and GABAa receptor antagonist were observed in this experimental study. Numerous histological and electrophysiological studies have been performed on the contribution of GABA to the auditory pathway; however, the spatio-temporal patterns of excitatory propagation and the relationships between GABA receptor and excitatory propagation have yet to be reported. Using an optical recording technique and a voltage-sensitive dye, the spatio-temporal patterns of excitatory propagation were observed in the auditory brainstem slices of early postnatal mice. A bath containing 50 microM GABA was applied, which largely inhibited the excitatory activities along the vestibulocochlear pathway. Bicuculline methiodide (BMI), a competitive antagonist against GABAa receptor, partially reversed the effects of GABA on the optical signals. Bath application of BMI alone helped to facilitate the depolarization course and its effect was apparent as an enlargement of the depolarized region from the cochlear nucleus and vestibular nucleus to some adjacent brainstem nuclei, as well as enhancing the amplitude of changes in the optical signals. The experimental results seem to suggest that GABAa receptors are widely distributed in an early postnatal auditory brainstem. GABA exhibited a greater modulating effect in the adjacent brainstem nuclei, which are involved in complex information processes, than that observed in the modulating primary auditory pathway. In the present experiment, significant GABAergic contributions to the optical recordings in the auditory brainstem were observed.     

Alpha-mangostin induces Ca2+-ATPase-dependent apoptosis via mitochondrial pathway in PC12 cells

We investigated the cell death effects of eight xanthones on PC12 rat pheochromocytoma cells. Among these compounds, alpha-mangostin, from the fruit hull of Garcinia mangostana L., had the most potent effect with the EC(50) value of 4 microM. Alpha-mangostin-treated PC12 cells demonstrated typical apoptotic DNA fragmentation and caspase-3 cleavage (equivalent to activation). The flow cytometric analysis indicated that this compound induced apoptosis in time-and concentration-dependent manners. Alpha-mangostin showed the features of the mitochondrial apoptotic pathway such as mitochondrial membrane depolarization and cytochrome c release. Furthermore, alpha-mangostin inhibited the sarco(endo)plasmic reticulum Ca(2+)-ATPase markedly. There was a correlation between the Ca(2+)-ATPase inhibitory effects and the apoptotic effects of the xanthone derivatives. On the other hand, c-Jun NH(2)-terminal kinase (JNK/SAPK), one of the signaling molecules of endoplasmic reticulum (ER) stress, was activated with alpha-mangostin treatment. These results suggest that alpha-mangostin inhibits Ca(2+)-ATPase to cause apoptosis through the mitochondrial pathway.     

Four new triterpenoids from Maytenus ilicifolia

Four new triterpenoids with various skeletons, maytefolins A-C (1-3) and uvaol-3-caffeate (4), were isolated from the leaves of a Brazilian medicinal plant, Maytenus ilicifolia, together with five known triterpenoids. Of these triterpenoids only erythrodiol exhibited significant cytotoxicity against KB/S, KB/VJ300, and KU 19-20 cells.     

Induction of CD28 on the new myeloma cell line MOLP-8 with t(11;14)(q13;q32) expressing delta/lambda type immunoglobulin

The novel multiple myeloma (MM) cell line MOLP-8 carrying the t(11;14) (q13;q32) was established from the peripheral blood of a 52-year-old Japanese male patient with Bence-Jones delta/lambda type MM (stage IIIA with hyperammonemia). The growth of MOLP-8 cells is constitutively independent of exogenous growth factors or feeder cells. MOLP-8 cells grow mainly as free floating single cells and slightly adherent on the bottom of the plastic culture flask. Wright-Giemsa-stained MOLP-8 cells show the typical plasma cell morphology with abundant cytoplasm, heterogeneous cell size and one to three nuclei. The immunoprofile of MOLP-8 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) delta/lambda chains, CD10, CD29, CD38, CD40, CD44, CD49b, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte-associated immunomarkers. CD28 became positive after co-culture of MOLP-8 cells with bone marrow adherent stromal (BST) feeder cells for a week. About 30% of MOLP-8 cells adhered strongly to the BST cells, but the cellular adhesion was clearly inhibited by addition of either anti-CD29 or anti-CD106 monoclonal antibody, suggesting a specific cellular adhesion through alpha4beta1-integrin-VCAM-1 interaction. The novel MOLP-8 cell line together with the present myeloma cell lines will present useful model systems in the investigation of the biology of MM.