A clustering of epidural abscesses in chronic hemodialysis patients: risks of salvaging access catheters in cases of infection.

The objective of this study was to investigate factors that might increase the risk of epidural abscesses in hemodialysis patients. The charts of all hemodialysis patients presenting with an epidural abscess over a period of 5 yr at Duke University Hospital and the Durham Veterans Administration Medical Center were reviewed for patient demographics, months on dialysis, vascular access, recently treated infections, signs and symptoms at presentation, and results of any surgical intervention. Ten patients developed an epidural abscess during a 5-yr period. Severe, debilitating back pain was the only consistent initial complaint. Eight patients had dual-lumen intravenous catheters for hemodialysis access, and five patients had or were receiving parenteral antibiotics for catheter salvage. There were no consistent physical, clinical, or laboratory findings. Surgical drainage of the abscess with removal of the hemodialysis catheters and parenteral antibiotics were required for cure in six patients. It was concluded that attempts at catheter salvage with parenteral antibiotics has significant risks for complications. Hemodialysis patients with recently treated or ongoing bacteremia who complain about severe and debilitating back pain with or without neurologic findings should raise the suspicion of an occult epidural abscess.

Clinical considerations in hemodialysis access infection.

Hemoaccess infections remain a substantial cause of morbidity in patients on hemodialysis, especially with the increasing reliance on prosthetic devices as the average age of the hemodialysis population increases. Access manipulation, either through needle puncture or secondary surgical procedures, is the primary etiology of infection. Other conditions such as access location, patient hygiene, and intravenous drug use can cause contamination. Local evidence of inflammation or infection, especially pain and purulence, are the most reliable signs of infection; however, the access can be infected and there may be minimal systemic symptoms. Medical therapy must be directed primarily against Staphylococcus aureus, with vancomycin being used most frequently. There are distinct conditions in which infection with gram-negative bacilli is also common. A coordinated effort between medical management and surgical intervention is essential to optimize therapy. Several situations, such as loss of vascular integrity or infection at anastomosis sites, mandate full excision of the graft. However, the access or at least the access site, can be preserved through creative surgical intervention along with aggressive medical treatment. Approaches to the diagnosis and treatment of infection in autologous arteriovenous fistulae, polytetrafluoroethylene arteriovenous conduits, and cuffed dual-lumen venous hemodialysis catheters are discussed.

Clinical characteristics of atherosclerotic renovascular disease.

In addition to its role in secondary hypertension, ischemic renal disease is becoming recognized as a significant cause of renal insufficiency. The prevalence and natural history of this disease remain unknown due to difficulty in identification of the process. There are several scenarios that may help alert the clinician to the presence of atherosclerotic renovascular disease. In hypertensive patients, poorly controlled blood pressure on several medications or rapid acceleration of hypertension can suggest renovascular disease. In addition, high-grade retinopathy or abdominal bruits seem to be associated with this condition: bruits have the highest positive predictive value of the clinical signs. Renal artery stenosis also may be related to rapidly progressive renal failure. The classic association is renal failure after use of angiotensin-converting enzyme inhibitors; however, acute renal failure induced through any sudden therapeutic decrease in blood pressure may imply the presence of renal artery stenosis. Unexplained azotemia in the elderly patient also has been associated with renovascular disease. It is possible that the majority of patients with renal artery stenosis are those with a clinically silent process. Advanced age, peripheral atherosclerotic vascular disease, and coronary artery disease may all have a high association with stenosis of one or both renal arteries. It is nevertheless unknown whether a radiographically detected lesion implies current or future clinical complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Causes of hemodialysis access failure.

Complications of hemodialysis access remain significant problems in the population receiving renal replacement therapy. The causes of access loss must be recognized before appropriate interventions can be designed. The native primary arteriovenous fistula is the access of choice because of its good survival characteristics and low rate of complications. Unfortunately, a substantial number of patients have vasculature insufficient to create and maintain this access. Once a primary fistula is established, thrombosis is the leading cause of failure: its causes can be divided into early (less than 6 weeks) and late complications. In patients unable to have fistulas, arteriovenous conduits of expanded polytetrafluoroethylene (ePTFE) are now the prostheses of choice. Again thrombosis is the leading cause of this access loss but there is also a substantial rate of failure from infection, pseudoaneurysms, perigraft hematomas, and simple attrition of the prosthesis. Thrombosis of ePTFE grafts is usually associated with anatomic stenosis at the venous anastomosis, within the graft itself, or in the central venous system. Graft thromboses cannot always be attributed to anatomic lesions: in these circumstances, thrombosis has been attributed to low-flow states. It is possible that the normal balance between endothelial procoagulant and anticoagulant forces are disrupted within the arteriovenous conduit. Recurrent venipuncture, hemodialysis therapy itself, or pathophysiological forces inherent in the access may all favor hemostasis. Infection also causes significant hemodialysis access morbidity. The causative organism is usually Staphylococcus aureus but several other pathogens have been reported. Ideally, all prostheses should be removed when infected, but this approach must be tempered by the reality of limited hemodialysis access sites. There is no consensus as to the best therapeutic approach to access infection.

Elevation of cAMP is required for down-regulation, but not agonist-induced desensitization, of endogenous dopamine D1 receptors in opossum kidney cells. Studies in cells that stably express a rat cAMP phosphodiesterase (rPDE3) cDNA.

D1 dopamine receptors stimulate cAMP accumulation in opossum kidney (OK) cells, but this response is attenuated by pretreatment with dopamine. Dopamine pretreatment also causes a reduction in D1 dopamine receptor number. We transfected OK cells with a rat cAMP phosphodiesterase cDNA (rPDE3) in order to determine the contribution of elevations of cAMP to those two phenomena. Wild-type (WT) OK cells were compared to three clones (C, H, and N) which demonstrated stable expression of the rPDE3 phenotype and genotype, rPDE3 RNA expression was confirmed in clones C, H, and N (but not in WT-OK cells) by reverse transcriptase-polymerase chain reaction. A functional rPDE3 phenotype was demonstrated in that dopamine-responsive cAMP accumulation was absent in clones C, H, and N in intact cells, but could be restored by preincubation with cAMP phosphodiesterase inhibitors, or by using washed membranes from those clones. All three clones had increased cAMP phosphodiesterase activity when compared to WT-OK cells (approximately 100% increase), and blunted or absent dopamine (1 microM)-induced protein kinase A activation. After pretreatment with dopamine (1 microM) for 1 h, clones C, H, and N desensitized equally well as WT-OK cells (approximately 40-50% reduction in maximal increase in cAMP). In contrast, down-regulation of D1 dopamine receptors was blunted for clone C (20% receptor loss) and absent for clones H and N, when compared to a 45% loss of receptors for WT-OK cells. These findings suggest that in OK cells pretreated with 1 microM dopamine (i) cAMP accumulation is not necessary for dopamine-induced desensitization, but (ii) is necessary for down-regulation of D1 dopamine receptors, and (iii) that the down-regulation and desensitization processes may be differentially regulated.

Thromboxane binding and signal transduction in rat glomerular mesangial cells.

Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (1R-[1 alpha(Z),2 beta,3 beta,5 alpha])-(+)-7-(5-[(1,1'-biphenyl)- 4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

8-hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells.

Bovine pulmonary artery smooth muscle (SM) cells express a novel 5-hydroxytryptamine (5-HT) (5-HT4-like) receptor coupled to cAMP accumulation. cAMP radioimmunoassay established the agonist and antagonist profiles of this receptor. 5-HT (EC50 = 91 +/- 33 nM) and 5-methoxytryptamine were equipotent at the SM cell 5-HT receptor and both were more potent than 5-carboxamidotryptamine. Other tryptamine derivatives were less potent but remained full agonists. These findings are consistent with previous reports regarding 5-HT4 and 5-HT4-like receptors in the central nervous system. The most potent antagonists were the antidepressant compounds nortriptyline (IC50 = 177 +/- 153 nM) and zimelidine (IC50 = 202 +/- 101 nM). The 5-HT3 and 5-HT4 antagonist 3-tropanyl-indole-3-carboxylate (ICS 205-930) was also a competitive antagonist at this 5-HT4-like receptor (pA2 = 6.3). Antagonist affinities differed slightly at the SM cell receptor, compared with other 5-HT4 and 5-HT4-like receptors in the central nervous system. Nonetheless, the SM cell 5-HT4-like receptor displayed the same differential antagonist potencies as reported for these other receptors (ICS 205-930 > MDL 72222 and mianserin > ketanserin). 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was the most potent agonist for this 5-HT4-like receptor (EC50 = 6.4 +/- 3.4 nM). 8-OH-DPAT-induced cAMP accumulation could be blocked by ICS 205-930 but not by the 5-HT1A antagonist 1-(2-methoxyphenyl)-4-[4-(2-pthalimido)butyl]piperazine hydrobromide, distinguishing the SM cell 5-HT receptor from 5-HT1A receptors. The mechanism of 5-HT-stimulated cAMP production was also investigated. First, GTP augmented basal and 5-HT-stimulated cAMP accumulation. Second, antisera to the carboxyl terminus of the alpha subunit of Gs, attenuated 5-HT-mediated adenylate cyclase activation. This established that 5-HT-stimulated cAMP accumulation in SM cells required GS. These findings suggest that SM cells express a novel 5-HT4-like receptor positively coupled to adenylate cyclase. An unexpected finding was that 8-OH-DPAT is a potent partial agonist. These studies suggest that there may be heterogeneity among 5-HT4-like receptors.

Functional expression of human 5-HT1A receptors and differential coupling to second messengers in CHO cells.

The signal transduction linkages of the cloned human 5-HT1A receptor as expressed stably in CHO cells were studied. A transfected clonal cell line which expresses 900 +/- 36 fmol 5-HT1A receptor/mg protein (designated CHO-5-HT1A/WT-27) responded to 5-HT and/or 8-OH-DPAT by coupling to several second messenger pathways. The 5-HT1A receptor inhibited, but did not stimulate, membrane adenylyl cyclase activity and whole cell cAMP accumulation in a dose-dependent manner (for 5-HT, IC50 = 146 +/- 27 and 55 +/- 12 nM, respectively). Activation of the receptor was associated with other signal transduction linkages: (i) a 40-50% increase in hydrolysis of inositol phosphates (for 5-HT, EC50 = 1.33 +/- 0.15 microM for 5-HT), (ii) a transient elevation of cytosolic Ca2+ levels (apparent at 1-100 microM 5-HT) which was not affected by chelation of extracellular Ca2+ by EGTA, and (iii) an augmentation of [3H]-arachidonic acid release pharmacologically with the calcium ionophore A23187 or by activation of endogenous thrombin or P2 purinergic receptors (for 5-HT, EC50 = 1.22 +/- 0.17 microM). This pathway may be an amplification mechanism for signaling in anatomic regions with high concentrations of several neuro-transmitters, hormones or autacoids, such as at neuronal junctions or near areas of platelet aggregation. All linkages were sensitive to pertussis toxin pre-treatment (IC50 approximately 0.5-0.6 ng/ml x 4.5 h for all pathways), suggesting the involvement of Gi protein(s) in these signal transduction pathways. Coupling to varied signal transduction pathways in a single cell system may be a common feature of receptors which classically inhibit adenylyl cyclase such as the 5-HT1A receptor.

Dendritic and synaptic properties of collicular neurons: a quantitative light and electron microscopical study of Golgi-impregnated cells.

The present study deals with a light- and electron microscopic morphometric analysis of Golgi-impregnated neurons in the superior colliculus of rats with the purpose to unravel inter- and intralaminar differences in their dendritic and synaptic organization. In particular, layer IV was studied and compared with its boundary layers III and V. The results show that collicular cells in layer IV basically form a homogeneous population with respect to the number of primary dendrites, the total length of impregnated dendrites, and the diameter, ellipticity, and orientation of dendritic fields and somata of Golgi-impregnated neurons. Somata of reconstructed small cells in layer III and IV as well as V have all a similar density of about 40 synaptic contacts per 100 microns2 surface. However, the cell bodies of large multipolar cells in layer V have a slightly but significantly larger synaptic density (about 50 per 100 microns2). Dendrites of large and small collicular cells had no significantly different synaptic densities (43 and 48 per 100 microns2, respectively). In conclusion, the present results show only minor dendritic and synaptic differences between individual cells in the same layer, as well as in neighboring layers, which implies a low degree of cellular and synaptic intra- and interlaminar differentiation. It is discussed that this organization differs markedly from that in other visual centers, including the collicular homologue, the tectum of lower vertebrates, and the mammalian visual cortex, where pronounced inter- and intralaminar differentiations exist. Such an organization may provide a framework of laminar specificity by which distinct cell types may select a restricted set of input out of all information available. The present quantitative investigation suggests that a similar framework is not present in the superior colliculus.

5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools.

Regulation of phosphate uptake was studied in a HeLa cell line after transfection with DNA encoding the human 5-HT1A receptor. In these cells, 5-HT stimulates sodium-dependent phosphate uptake via protein kinase C activation. Endogenous histamine H1 receptors (739 +/- 20 fmol/mg protein) were identified with [3H]pyrilamine. Histamine (i) stimulated phosphoinositide hydrolysis (EC50 = 8.6 +/- 4.1 microM), (ii) activated protein kinase C (2.4-fold increase in activity), and (iii) increased phosphate uptake (EC50 = 3.2 +/- 1.8 microM) by increasing maximal transport (Vmax(basal) = 6.2 +/- 0.3 versus Vmax(histamine) = 9.1 +/- 0.4) without changing the affinity of the transport process for phosphate. Prolonged treatment with 16 microM phorbol 12-myristate 13-acetate completely blocked protein kinase C activation and markedly attenuated the stimulation of phosphate uptake induced by histamine, establishing that 5-HT and histamine stimulate phosphate uptake through the common pathway of protein kinase C activation. The linkages of the histamine H1 and 5-HT1A receptors to G protein pools were assessed in two ways. (i) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake associated with histamine were insensitive to pertussis toxin, whereas those associated with 5-HT were very sensitive to pertussis toxin. (ii) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake induced by histamine and 5-HT were additive. These findings suggest that distinct receptor types can stimulate phosphoinositide hydrolysis, protein kinase C, and phosphate uptake in an additive fashion through distinct pools of G proteins in a single cell type.

Thapsigargin demonstrates calcium-dependent regulation of phosphate uptake in HeLa cells.

We used thapsigargin, a sesquiterpene lactone that mobilizes intracellular Ca without increases in inositol phosphates or major activation of protein kinase C (PKC), to test the specific effects of increasing cytosolic Ca on Na-dependent phosphate uptake in HeLa cells. Thapsigargin increased the Vmax for phosphate uptake from 5.40 +/- 0.26 to 7.86 +/- 0.43 nmol.mg protein-1.3 min-1 (n = 7, P less than 0.001) without change in the apparent Km for phosphate, which averaged 0.15 +/- 0.02 mM. The effect of thapsigargin was dependent on concentration and time. Inactivation of PKC by overnight exposure to 16 microM phorbol 12,13-dibutyrate did not eliminate the effect of thapsigargin, although it completely abolished the effects of phorbol ester on phosphate uptake. Thus thapsigargin are not dependent on PKC. As in other cell systems, thapsigargin increased cytosolic Ca concentration. Removal of extracellular Ca diminished the increase in cytosolic Ca and eliminated the effect of thapsigargin on phosphate uptake. Collectively, our data indicate that Na-dependent phosphate uptake in HeLa cells can be regulated by at least three specific signaling pathways: protein kinase A, PKC, and increased cytosolic Ca.

Synaptic morphometry and synapse-to-neuron ratios in the superior colliculus of albino rats.

The superior colliculus of mammals is generally divided into seven layers on the basis of the distribution of myelinated fibers, which are densely packed in layers III, V, and VII but sparse in the other layers. The laminar distribution of afferents and efferents allows, in addition, for the distinction of a superficial visual zone (layers I-III) and a deeper multimodal and premotor zone (layers IV-VII). Collicular neurons, however, do not show a lamination pattern, but are rather homogeneously distributed with only gradual transitions (Albers et al.: J. Comp. Neurol. 274:357-370, '88). The present study analyses whether the distribution of collicular synapses is correlated with the laminar organization of collicular axons or rather with the more homogeneous distribution of collicular neurons. For this purpose, the size and density of synaptic terminals and contacts as well as synapse-to-neuron ratios were determined in all collicular layers of albino rats by means of quantitative analysis of electron microscopic pictures. The size of presynaptic terminals and contacts does not differ significantly between individual collicular layers. On average, presynaptic terminal diameter is 1,079 nm, and synaptic contact size 338 nm, while 23% of all contacts are of the symmetrical type with pleiomorphic vesicles. The average numerical synaptic density is 422 million per mm3. This value is significantly higher in layers I and II (on average 670 million per mm3) than in layers III-VII (on average 370 million per mm3). The synapse-to-neuron (S/N) ratios calculated show that collicular neurons have on average 6,120 synaptic contacts on their receptive surface. The S/N ratio is lowest in layer III (4,330), while this ratio is highest in layers I and VII (i.e., 8,970 and 8,560 respectively). Layer II has a significantly higher S/N ratio than layer III (i.e., 8,060 and 4,330, respectively). Our results show that the size of synaptic terminals and contacts is not correlated with the different connectivity patterns of the distinct collicular layers. However, the density of synapses as well as the synapse-to-neuron ratios show a certain degree of laminar differentiation. In particular the superficial visual zone appears to be inhomogeneous in this respect, since layers I and II have a significantly higher density of synapses and higher S/N ratios than layer III. The deeper collicular zone is more homogeneously organized with synaptic densities similar to that of layer III and gradually increasing synapse-to-neuron ratios from layer IV to layer VII.

Morphometric parameters of the superior colliculus of albino and pigmented rats.

The superior colliculus (SC) or optic tectum of mammals consists of seven layers, numbered I-VII from superficial to deep, each of which has distinct connectivity patterns and electrophysiological response properties. The present study is devoted to a morphometrical analysis of neuronal diameters, densities, and numbers in different layers and regions of the SC of albino as well as pigmented rats in order to present a quantitative characterization of the collicular neuronal population involved in the different connectivities and functions of these compartments. The morphometric parameters were calculated from tracings of nuclei and cell bodies by means of Kontron-Videoplan equipment and a Micro PDP 11/23 computer. The mean soma diameter per superior colliculus appears to be 12.0 microns, the average neuronal density 70 cells per 0.001 mm3, and the total number of neurons about 600,000. The mean soma diameter gradually increases from superficial to deep layers (i.e., from 10.0 to 14.0 microns). Cellular density is highest in layer III, the retinal afferent layer (90 cells per 0.001 mm3), and decreases both in more superficial layers (to about 80 in layer I) and deeper layers (to about 44 in layer VII). About 25% of all collicular neurons are situated in layer II whereas layer I contains the lowest percentage of cells (4%). Rostrally within each collicular layer, cellular volumes are about 25% larger than caudally. On the other hand, neuronal densities are rostrally about 38% lower than caudally in all layers except for layers VI and VII. We conclude that collicular neurons, in contrast to collicular axons, are not arranged in distinct layers or clusters but basically establish a random network with only gradual transitions. In this respect, no statistically significant differences were observed between albino and pigmented rats.

Potassium ferricyanide: a stabilizer for horseradish peroxidase.

Sodium nitroprusside has been used as a stabilizing agent for HRP histochemistry since 1919. However, it is known to have toxic effects orally, intravenously, and subcutaneously. In order to avoid the use of sodium nitroprusside as a stabilizer in HRP histochemistry, we have tested other chemically related compounds to stabilize the reaction product equally well. We will show that potassium ferricyanide is an excellent stabilizer of the chromogen reaction product. In addition, the reaction product remains stable without noticeable changes over a period of several months. As it is far less toxic than sodium nitroprusside, it should be the stabilizer of choice, especially in those laboratories where the histochemical HRP reaction is used frequently.