Role of variants rs5030717 and rs5030718 of TLR4 in the risk prediction of nephropathy, hypertension and dyslipidaemia in type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus describes a metabolic disorder characterised by prolonged elevated blood glucose that brings a risk of developing microvascular and macrovascular disease. Several factors, such as dysregulation of the Toll-like receptor 4 (TLR-4), are reputed to contribute to the multiple pathophysiological disturbances responsible for impaired glucose homeostasis. We hypothesised that variants rs5030717 and rs5030718 of TLR4 are associated with diabetic nephropathy, hypertension and dyslipidaemia. MATERIAL & METHODS: We recruited 370 diabetics (122 with nephropathy, 119 with hypertension and 129 with dyslipidaemia) and 120 ethnicity matched healthy controls. TLR4 polymorphisms were evaluated using polymerase chain reaction followed by restriction fragment length polymorphism analysis. The genotyping data were compared between cases and controls using chi-square test and logistic regression analysis. RESULTS: Although there was no overall difference in the genotype frequencies of TLR4 rs5030717 in diabetes v controls, the genotype frequencies of diabetic dyslipidaemia cases compared with controls were different (p = 0.001). Overall, the rs5030718 GA and GG genotype frequencies in the entire diabetes cohort were different from those of the controls (p = 0.037), and the frequencies of diabetic nephropathy cases (p = 0.03) and diabetic dyslipidaemia cases were different (p = 0.001) compared with controls. There were no links with diabetic hypertension. CONCLUSION: TLR4 polymorphisms rs5030717 and rs5030718 may be useful in predicting those type 2 diabetics who are at risk of hypertension, nephropathy and/or dyslipidaemia.

The effect of aging on pars compacta of the substantia nigra in rhesus monkey.

This aim of this study was to re-evaluate the types of neurons present in pars compacta of the substantia nigra in the rhesus monkey, and then to determine the effects of aging on the morphology of both the neurons and neuroglial cells. The substantia nigra was therefore examined in Golgi impregnated material, in sections labeled with antibodies to tyrosine hydroxylase and to GABA, and in tissue embedded in plastic for light and electron microscopy. Three types of neurons were encountered: (1) large multipolar neurons with prominent Nissl bodies, (2) bipolar neurons that are medium sized and spindle-shaped, with Nissl bodies confined to the poles of the deeply indented nuclear envelopes, and (3) small multipolar cells with sparse Nissl substance. The large and medium neurons are believed to be dopaminergic and the small neurons GABA-ergic. With age, all of the neurons accumulate lipofuscin, especially the small multipolar neurons. In addition, Marinesco bodies appear within the nuclei of the large multipolar neurons. The dendrites are most severely affected by age; many of them lose organelles and their cytoplasm can become filled with vacuoles, membranous whorls, and dense bodies. In old monkeys many of the astrocytes have inclusions and in the neuropil there is a striking increase in the number of astrocytic processes. Also large spheroids appear. These appear to be derived from astrocytes and they have a core of dense granular material surrounded by a paler peripheral zone of cytoplasm from which processes can extend. Most of the oligodendrocytes have dense inclusions in their cytoplasm and many of the myelin sheaths break down. Microglial cells can become enormously swollen by phagocytosed material. Although both neurons and neuroglial cells are affected by age, no entities that could be construed to be dying neurons were encountered.

Age-related neuronal loss from the substantia nigra-pars compacta and ventral tegmental area of the rhesus monkey.

This study was undertaken to document the effect of age on the volume, number, and size of neurons in the substantia nigra pars compacta (SNpc), the paranigral (VTApn), and the parabrachial pigmentosus (VTApbp) nuclei of the VTA in behaviorally well-characterized rhesus monkeys. Using a point counting technique and unbiased stereological methods, we found no significant effects of age on the volume of these dopaminergic brain stem nuclei, but there was significant age-related loss of total number of neurons in the SNpc and VTApn. The loss of neurons in the SNpc was found to be greater for neurons measuring less than 200 microm2 in cross sectional area, a size corresponding to the small GABAergic neurons in this nucleus. Age-related loss of total number of neurons in the SNpc and VTApn showed significant correlations with impairment on the delay phase of the delayed non match to sample test (DNMS), in the VTApn with impaired performance on the delayed recognition span test (DRST), and in the SNpc with impaired performance on the spatial component of reversal tasks. These correlations suggest that the loss of neurons in the SNpc and VTApn in these old monkeys may contribute to these behavioral deficits.

Improving patient selection for coagulopathy testing in the setting of acute ischemic stroke.

To improve patient selection for specialized coagulation testing in the setting of ischemic stroke, the authors sought to identify factors associated with the presence of hypercoagulable states. Of 208 patients with ischemic stroke tested, undetermined stroke subtype was significantly associated with the presence of coagulopathy, but only 60% were treated with warfarin. The frequency of coagulopathy in selected patients with ischemic stroke (5%) is low, and establishing the diagnosis did not uniformly influence treatment.

Use of specialized coagulation testing in the evaluation of patients with acute ischemic stroke.

OBJECTIVE: To investigate the use and appropriateness of specialized coagulation tests in the evaluation of patients with acute ischemic stroke and identify factors that influence test ordering. BACKGROUND: Coagulation abnormalities are a rare but recognized cause of ischemic stroke. METHODS: Patient demographics, stroke risk factors, history of venous thrombosis or miscarriage, family history of stroke, and the results of specialized tests for coagulation disorders were recorded for a consecutive series of ischemic stroke patients over age 18 admitted to an academic medical center over 3 years (n = 674). Factors associated with testing were identified with univariate analyses in a random sample of two-thirds of the patients (n = 450). Multivariate logistic regression modeling was then used to identify variables independently associated with testing and then validated in the remaining patients (n = 224). RESULTS: Of the 31% of patients (n = 208) tested for coagulopathies, 29% (n = 60) were tested when the result was unlikely to influence therapeutic decisions. Historical factors associated with an increased incidence of a coagulopathy, such as history of venous thrombosis or miscarriage, were not commonly documented. The absence of small-artery atherosclerosis (OR 0.36, 95% CI 0.17 to 0.76) and younger age (OR 0.89 per year, 95% CI 0.87 to 0.92) were independently related to the frequency of specialized coagulation testing. CONCLUSIONS: One-third of specialized coagulation tests were ordered when the test results were unlikely to affect therapeutic decisions. Age was the only clinical factor increasing the likelihood of a coagulopathy that appeared to influence ordering of specialized coagulation tests.

The effect of mannitol on intracranial pressure in relation to serum osmolality in a cat model of cerebral edema.

OBJECTIVE: To determine whether intravenous mannitol administration reduces intracranial pressure (ICP) in a cat model of brain edema by changing serum osmolality. DESIGN: Prospective, controlled study. SETTING: Pediatric intensive care unit laboratory in a university hospital. INTERVENTIONS: Intraparenchymal ICP monitors were placed in 12 adult cats which subsequently underwent 60 min of continuous arteriovenous hemofiltration with countercurrent dialysis (CAVH-D), using sterile water with potassium chloride as a dialysate. The ultrafiltrate was replaced with a hypotonic solution causing a rapid reduction in serum osmolality while maintaining a euvolemic state. In six cats (control group) no further interventions were instituted, while in the six other cats (mannitol group) 1g/kg mannitol was administered intravenously immediately after CAVH-D had been discontinued. ICP was monitored continuously, and serum osmolality was determined at 15-min intervals during CAVH-D and for 30 min thereafter. RESULTS: ICP increased significantly in both the control and mannitol groups during 60 min of CAVH-D. After CAVH-D, ICP was reduced in the mannitol group while ICP remained significantly higher in the control group. An inverse linear correlation was demonstrated between serum osmolality and ICP values in the control group throughout the experiment, as well as during the first 60 min in the mannitol group. However, no such correlation existed in the mannitol group after mannitol administration, as no significant changes in serum osmolality were observed while a marked reduction in ICP values occurred. CONCLUSION: Mannitol is effective in reducing increased ICP in this model of euvolemic brain edema. However, 15 min after mannitol administration, no relationship between a continued decrease in ICP and a change in serum osmolality could be established. We postulate that the beneficial effect on ICP by mannitol outlasts its possible instantaneous and short-lived effect on serum osmolality.

Reduction of baroreceptor-related sympathetic discharge during NMDA receptor antagonism.

N-Methyl-D-aspartate receptor blockade by MK-801 produced changes in the baroreceptor related component of pre-ganglionic sympathetic discharges in kittens. Spectral analyses carried out on sympathetic and blood pressure signals demonstrated: (a) a marked reduction in amplitude of peaks, 2-6 Hz range, in sympathetic autopower spectra and (b) a substantial decrease of coherence between sympathetic and blood pressure signals in the 2-6 Hz range.

Effects of bicuculline on inspiratory activities in piglets.

Antagonism of the alpha receptor sub-type at gamma-aminobutyric acid (GABA) recognition sites in developing pigs was evaluated using the GABAA receptor antagonist bicuculline. The effect of bicuculline infusions was to produce an increase of phrenic and hypoglossal discharge amplitudes. This bicuculline-induced effect on discharge amplitude was manifested in autopower spectra as an increase in the power of peaks located in the medium-frequency (10-50 Hz) band. More importantly, coherence estimates were increased by bicuculline administration demonstrating GABA-mediated influences on a central pattern generator with output in the 10-50 Hz band.

Evidence for central patterning of sympathetic discharge in kittens.

Cervical sympathetic nerve discharges in kittens were examined using power spectral techniques to describe the frequency components of nerve activities and to obtain evidence of modulation by central rhythm generators. Peaks in sympathetic spectra appeared in the 4-11 Hz band; however, coherences between sympathetic nerves occurred at 4-5 Hz, while that between sympathetic and phrenic nerves occurred at 8-9 Hz. These findings indicate modulation by sympathetic and inspiratory rhythm generators.

The effects of N-methyl-D-aspartate antagonism on the inspiratory activities of developing animals.

In developing kittens, antagonism of N-methyl-D-aspartate receptors by systemic injections of MK-801 produced: (a) blockage of the inspiratory off-switch, prolonging phrenic and hypoglossal discharges; (b) age-dependent changes of sympathetic inspiratory discharges, elimination of inspiratory-related discharges in animals less than or equal to 36 days old and prolongation of these discharges in older animals; (c) suppression of high-frequency oscillations in phrenic activities.

Effects of GABAA receptor antagonism on inspiratory activities in kittens.

In anesthetized kittens (< 1 to 24 days old), the effects of GABAA receptor antagonism on phrenic, hypoglossal and cervical sympathetic discharges were examined by i.v. bicuculline infusions during hyperoxia and hypoxia. Administration of bicuculline during hyperoxia produced marked increases in the amplitudes of inspiratory nerve discharges. During hypoxic stimulation (10% O2), the amplitudes of inspiratory activities decreased towards or below those observed during hyperoxia; bicuculline reversed this depression and restored inspiratory discharges. Our results indicated that GABAA receptors were functional shortly after birth, acting to mediate influences shaping inspiratory activity during hyperoxic breathing and during conditions of increased chemical drive.

Mycophenolate mofetil in myasthenia gravis: the unanswered question.

Myasthenia gravis (MG) is an autoimmune disease that leads to muscular weakness, which can significantly affect the patient's daily functions. If left untreated, the mortality rate can be as high as 30%. Effective immunosuppression is the cornerstone of treatment of MG, although most currently available immunomodulatory drugs are associated with unacceptable side effects, delayed onset of therapeutic action, or both. Mycophenolate mofetil (MMF) might be better tolerated than other immunosuppressants and many case reports and uncontrolled trials have indicated that it is effective in MG. However, two recently concluded clinical trials failed to demonstrate the efficacy of MMF in MG. This paper critically reviews the existing evidence on the efficacy of MMF in MG and provides the authors' view of its role in current practice.

An international, phase III, randomized trial of mycophenolate mofetil in myasthenia gravis.

BACKGROUND: This prospective, randomized, double-blind, placebo-controlled, phase III trial assessed the efficacy, safety, and tolerability of mycophenolate mofetil (MMF) as a steroid-sparing agent in patients with myasthenia gravis (MG). METHODS: Patients with acetylcholine receptor antibody-positive class II-IVa MG (MG Foundation of America [MGFA] criteria) taking corticosteroids for at least 4 weeks were randomized to MMF (2 g/day) or placebo for 36 weeks. The primary endpoint was a composite measure defined as achievement of minimal manifestations or pharmacologic remission (MGFA post-intervention status), with reduction of corticosteroid dose on a set schedule. Secondary endpoints included disease severity, quality-of-life scores, and safety. RESULTS: A total of 44% of MMF-treated (n = 88) and 39% of placebo-receiving (n = 88) patients achieved the primary endpoint (p = 0.541). Improvements in mean quantitative MG, MG activities of daily living, and 36-item Short-Form health survey scores were similar in both groups. Numbers of adverse events were similar in both groups. The most commonly reported adverse events in the MMF-treated group were headache (12.5%) and worsening of MG (11.4%), and in the placebo group, worsening of MG (20.5%) and diarrhea (10.2%). CONCLUSIONS: Initiation of mycophenolate mofetil (MMF) treatment was not superior to placebo in maintaining myasthenia gravis (MG) control during a 36-week schedule of prednisone tapering. There were no significant differences in the primary or secondary endpoints between the study groups. MMF was well tolerated and adverse events were consistent with previous studies. Experience from this large, international, multicenter, phase III study employing full MG Foundation of America guidelines will aid the design of future MG studies.

Respiration-related features of sympathetic discharges in the developing kitten.

The objectives of the present study were to identify sympathetic discharge features, either spontaneous or evoked, which exhibited maturational changes. The spontaneous sympathetic nerve discharge of all kittens was comprised of tonic activity as well as phasic inspiratory activity; the latter occurred synchronously with the C5 phrenic discharge. Sympathetic activity during inspiration was depressed (inhibited or disfacilitated) by vagal afferent inputs as was indicated by the marked increases of amplitude following vagotomy. Hypoxia (10% O2) elicited changes in the amplitude of the sympathetic inspiration-related discharge which were identical to those of the phrenic; discharge amplitudes were either increased, or decreased, or increased and then decreased (i.e. biphasic response). Regardless of the change in sympathetic inspiration-related activity during hypoxia, the amplitude of tonic activity during expiration was often increased. Such changes in the discharge characteristics of the cervical sympathetic nerve in response to vagotomy or hypoxia were not dependent upon maturational processes as they were similar to those of adult cats. However, an age-dependent phenomenon was revealed by autopower spectral analyses which showed that the phrenic and sympathetic nerve activities in animals > or = 45 day old were modulated by inputs from a common source because their coherence spectra exhibited correlated periodicities in the 7-10 Hz range.

Rapid, selective and direct spectrophotometeric determination of aliphatic amines with m-dinitrobenzene.

Color reaction has been studied for identification and spectrophotometric determination of aliphatic amines at room temperature by m-dinitrobenzene (m-DNB) as reagent. The lambda(max) value ranges from 458 to 570 nm. This is a simple and rapid method for determination of aliphatic amines in the acidic, water and acetone medium. Beer's law is verified for methylamine, dimethylamine, trimethylamine and n-butylamine in the range of 0.5-8 mg l(-1). The effect of pH on the molar absorptivity is investigated for a representative primary amine i.e. methylamine and it was observed that molar absorptivity increases from acidic to basic pH, with a sharp increase at pH 12. The kinetic of reaction was also studied and found that reaction time has marked effect on the molar absorptivity of electron donor-acceptor (EDA) complex. The detection of methyl amine has been reported in three real samples of water.

Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts.

Amino acid substitutions in collagen that impair folding of the triple helix result in significant increases in intracellular degradation of newly synthesized collagen. We have studied the effects of agents that cause other kinds of defects in collagen: hydroxynorvaline, a threonine analog that interferes with association of pro-alpha chains; and puromycin, an antibiotic that causes premature release of nascent polypeptides. cis-Hydroxyproline and cycloheximide, whose effects on collagen synthesis and degradation have already been studied and reported, were employed as reference compounds. Human fetal lung fibroblasts were used in these experiments. All the agents inhibited total protein production, and all except cycloheximide inhibited percentage collagen production. Intracellular collagen degradation was increased in cultures exposed to puromycin, hydroxynorvaline, and cis-hydroxyproline, but not in cultures exposed to cycloheximide. These results suggest that pro-alpha chains that were either unassociated (due to hydroxynorvaline) or shortened (due to puromycin) were recognized as abnormal and degraded to the same extent as chains that contained cis-hydroxyproline. However, the increases in degradation could not account completely for the decreases in collagen production (except when cis-hydroxyproline was used at low concentrations). These findings indicate that, in addition to rendering newly synthesized procollagen molecules or partial polypeptide chains more susceptible to intracellular degradation, puromycin, hydroxynorvaline, and cis-hydroxyproline significantly inhibited collagen synthesis.

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts.

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.

Fetal type 2 pneumocytes form alveolar-like structures and maintain long-term differentiation on extracellular matrix.

We investigated the effects of reconstituted basement membrane (a crude extract of the Engelbreth-Holm-Swarm tumor) on type 2 pneumocyte differentiation during long-term culture. Cells were derived from mature 29 d fetal rabbits. Morphology was studied by light and electron microscopy. On thin gel, the cells initially segregated into clumps; they were cuboidal with apical microvilli and contained lamellar bodies, but dedifferentiated by 8 d. On thick gel, epithelial cells associated into spherical clusters surrounding a central lumen. These alveolarlike structures persisted at least 22 d. The cells were cuboidal and had lamellar bodies and intercellular tight junctions; they exhibited polarity, with apical microvilli facing the lumen, basally located nuclei, and gel matrix abutting the basal surface. In contrast, cells cultured on plastic formed colonies, then a monolayer, but dedifferentiated 5-7 d after plating. [14C]Acetate was used to label newly synthesized phospholipids. The amount of disaturated phosphatidylcholine (DSPC), expressed as a percentage of total phosphatidylcholine (PC), was used as an indicator of surfactant lipid production; percentage DSPC synthesized by cells cultured on thick gel did not change significantly, from 55 +/- 3 at 3 d, to 63 +/- 2 at 22 d in culture. DSPC synthesized by cells cultured on plastic decreased from 57 +/- 1% at 3 d to 45 +/- 2% at 22 d (p less than 0.001), which is consistent with the morphologic evidence of dedifferentiation. Synthesis of total PC compared with total phospholipid did not vary with either time in culture or substrate. This study emphasizes the importance of a complex extracellular matrix for maintenance of type 2 pneumocyte differentiation. The system should prove useful for studying the interaction of these cells with basement membrane, including the role of events occurring at the cell surface in modulating expression of a differentiated phenotype.

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts.

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.