Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation.

Monitoring of gene and protein expression in peripheral blood cells has significant potential for improving the diagnosis and therapy of many human diseases. As genomic-scale microarray and proteomic technologies are applied to peripheral blood, it is important to consider the variables that may affect interpretation of data. Here we report experiments performed to identify genes that are particularly sensitive to ex vivo handling prior to RNA extraction for gene expression microarrays or quantitative real-time RT-PCR assays. We examined Affymetrix gene expression in samples from eight normal individuals where blood was processed for RNA either immediately after blood draw or the next day following overnight incubation. These studies identified hundreds of genes that are sensitive to ex vivo handling of blood, and suggest that this is an important variable to consider when designing and interpreting human PBMC experiments.

Changes in behavioural contingencies produce a loss of tolerance to amphetamine hypophagia in rats despite continued feeding tests while drugged.

Rats administered amphetamine prior to access to milk in bottles develop tolerance to the hypophagic effect of the drug by learning to suppress stereotyped behaviours that interfere with feeding. When tolerant rats are later allowed to drink milk from a bottle in an unintoxicated state, tolerance is lost, even when drug exposure is held constant by administration of the drug after the test. In the present experiment, we show that tolerance can also be lost in the face of continued administration of amphetamine prior to milk tests, as a result of changes in the contingencies of reinforcement that govern the suppression of stereotypy. Rats were injected with 2 mg/kg amphetamine and given access to milk in bottles for 16 trials. Tolerance to the hypophagic effect was confirmed by dose-response tests in which milk was available in bottles. The rats were then injected with 2 mg/kg amphetamine prior to intraoral milk infusions for 21 trials. This method of feeding did not require the suppression of stereotypy to obtain milk. Subsequent dose-response tests in which milk was again presented in bottles revealed that tolerance was lost, even though intoxicated feedings were never interrupted. These results demonstrate that the contingencies of reinforcement governing the suppression of stereotypy determine whether tolerance is retained or lost.

Practice effects on coordination and control, metabolic energy expenditure, and muscle activation.

One defining characteristic of skilled motor performance is the ability to complete the task with minimum energy expenditure. This experiment was designed to examine practice effects on coordination and control, metabolic energy expenditure, and muscle activation. Participants rowed an ergometer at 100 W for ten 16-min sessions. Oxygen consumption and perceived exertion (central and peripheral) declined significantly with practice and movement economy improved (reliably) by 9%. There was an associated but non-significant reduction in heart rate. Stroke rate decreased significantly. Peak forces applied to the ergometer handle were significantly less variable following practice and increased stability of the post-practice movement pattern was also revealed in more tightly clustered plots of hip velocity against horizontal displacement. Over practice trials muscle activation decreased, as revealed in integrated EMG data from the vastus lateralis and biceps brachii, and coherence analysis revealed the muscle activation patterns became more tightly coordinated. The results showed that practice reduced the metabolic energy cost of performance and practice-related refinements to coordination and control were also associated with significant reductions in muscle activation.

Long-term retention of tolerance to amphetamine hypophagia following cessation of drug injections and feeding tests.

According to the instrumental learning model, tolerance to amphetamine hypophagia involves learning to suppress stereotyped movements that interfere with feeding. If both drug injections and feeding tests are then suspended, learning should be retained and no loss of tolerance should occur. However, previous studies have only assessed the retention of tolerance for 3-4 weeks. In the present study, retention intervals of 4-31 weeks were used. Rats were given daily injections of amphetamine (2 mg/kg) and access to milk for 30 min until tolerance developed to drug-induced hypophagia. Yoked controls were injected with saline. Both before and after this phase, dose-response (DR) tests were conducted. Drug injections and feeding tests were then suspended. At 4, 10, 18, and 31 weeks, both groups were injected with 2 mg/kg amphetamine and given access to milk for 30 min to assess the retention of tolerance. A final DR determination was then conducted. Most (88%) rats retained tolerance to 2 mg/kg amphetamine for 31 weeks. However, DR tests revealed that tolerance was not retained at 4 mg/kg. The results demonstrate that learned tolerance to amphetamine can be retained over long intervals when both drug injections and feeding tests are suspended.

Pseudohyperkalemia secondary to postsplenectomy thrombocytosis.

Hyperkalemia is a regularly encountered electrolyte abnormality. Less commonly recognized is pseudohyperkalemia. In vitro clotting results in the release of potassium from the formed elements of blood; this falsely elevates the serum potassium concentration. Usually attributed to thrombocytosis, leukocytosis, or hemolysis it is rarely reported in the postsplenectomy trauma patient. A 49-year-old man underwent splenectomy for a grade IV splenic injury. He developed an elevated serum potassium on postoperative day 16. His renal function remained normal, and an active search for causes of hyperkalemia failed to delineate a source for his elevated potassium. Pseudohyperkalemia was proposed as an etiology and confirmed by analyzing simultaneous serum (5.9 mEq/L) and plasma potassium levels (3.9 mEq/L). The serum potassium showed a concomitant rise with the evolution of the patient's postsplenectomy thrombocytosis. This is a case report of our patient and a literature review of this rarely reported and underestimated cause of a potentially serious electrolyte abnormality. We found through our patient that pseudohyperkalemia does occur in the postsplenectomy population and that this should be included in the differential diagnosis of any patient with an elevated serum potassium level and thrombocytosis.

Traumatic carotid artery dissection: a significant incidental finding.

Blunt traumatic carotid artery dissection remains controversial in terms of diagnostic screening, reported incidence, and management. Treatment options include observation, anticoagulation and endovascular stenting, and aggressive surgical repair of the carotid artery injury. Blunt traumatic carotid artery dissections were reviewed through a retrospective study of trauma registry records. Seven patients were identified from 3342 patients over 3 years. Six patients were identified incidentally during magnetic resonance imaging (MRI) cervical spine/brain screening and one patient during angiographic evaluation for possible penetrating neck injury without MRI/magnetic resonance angiography (MRA). A total of 189 patients underwent MRI screening over this 3-year period, demonstrating a relative incidence of 3.7 per cent, contrasting with the reported incidence of 0.08 to 0.4 per cent for all trauma patients. All seven patients suffered severe head injuries (mean Glasgow Coma Score = 4.7) requiring mean intensive care unit and hospital stays of 15.6 and 23.7 days, respectively. None of the patients showed evidence of stroke with CT scanning on presentation. None of the patients demonstrated clinical focal neurologic signs or symptoms indicating carotid injury or stroke. Six patients survived their acute trauma and were discharged to rehabilitation after initiation of observation (one patient) or anticoagulation (five patients). All six patients showed neurological improvement without deterioration clinically or radiographically. In conclusion we propose early aggressive screening through MRI/MRA of severely injured patients to detect occult carotid artery dissections. Conservative medical treatment for this occult injury has been effective in this series of patients.

Loss of tolerance to amphetamine-induced hypophagia in rats: homeostatic readjustment vs. instrumental learning.

According to the homeostatic model, the loss of tolerance to amphetamine-induced hypophagia requires a period of unrestricted feeding in the drug-free state, which transforms the compensatory response mediating tolerance ("hyperhunger") into a functional disturbance to homeostasis. In the absence of such a disturbance, tolerance should be retained. To test this prediction, rats tolerant to amphetamine's hypophagic effect were given a 4-week tolerance retention period during which milk intakes were restricted and deprivation levels held relatively constant. During this period the rats were assigned to one of the following drug treatment conditions: 1) saline injections both before and after daily milk tests (saline group); 2) saline injections before, and amphetamine injections after, daily milk tests (after group); 3) no injections and no milk tests (no-treatment group); or 4) amphetamine injections before, and saline injections after, milk tests (before group). Despite the restricted feeding regimen, both the saline and after groups lost tolerance. These results do not support the homeostatic model, but are consistent with the instrumental learning model, which views drinking milk in the undrugged state as analogous to receiving noncontingent reinforcement.

Differential roles of the tandem C1 domains of protein kinase C delta in the biphasic down-regulation induced by bryostatin 1.

Bryostatin 1 (Bryo), currently in clinical trials, has been shown to induce a biphasic concentration-response curve for down-regulating protein kinase C (PKC) delta, with protection of the enzyme from down-regulation at high Bryo doses. In our ongoing studies to identify the basis for this unique behavior of PKCdelta, we examined the participation of the two ligand binding sites (C1a and C1b) in the regulatory domain of the enzyme. Three mutants of PKCdelta prepared by introducing a point mutation in either C1a or Clb or both C1a and Clb were overexpressed in NIH 3T3 cells. All of the constructs retained a biphasic response to down-regulation assessed after 24-h treatment with Bryo. However, the roles of the individual C1 domains were different for the two phases of the response. For down-regulation, both the C1a and the C1b mutants displayed equivalent 3-4-fold reductions in their affinities for the ligand. For protection from down-regulation, a reduced protection was observed for the C1a mutant, which showed a broader biphasic curve compared with those for wild-type PKCdelta and the Clb mutant. Like wild-type PKCdelta, all of the mutants showed the same subcellular partitioning of the protected enzyme to the particulate fraction of the cells, arguing against changes in sensitivity to Bryo due to differences in localization. Likewise, relatively similar patterns of localization were observed using green fluorescent protein-PKCdelta constructs. We conclude that the C1 domains of PKCdelta do not have equivalent roles in inducing protection against Bryo-induced down-regulation. The C1a domain plays a critical role in conferring the degree of protection at high concentrations of Bryo. Elucidation of the differential effect of Bryo on PKCdelta may suggest strategies for the design of novel ligands with Bryo-like activities.

HIV infection among rural trauma patients: a blind serosurvey and literature review.

Human immunodeficiency virus (HIV) seroprevalence rates among rural trauma patients range between 0.15 and 1.32 per cent. A random sample of trauma patients treated at our rural trauma center between September 1994 and November 1995 was enrolled into a blind HIV serosurvey. Five hundred sixty-six of 1315 trauma patients (43%) were tested. Two of the 566 patients (0.35%) were HIV positive. A review of aggregate data for HIV infection among rural trauma patients in the United States show that 28 of the 4639 patients (0.60%) are HIV positive. We conclude that there was a low HIV incidence among our trauma patients from September 1994 to November 1995, and the cost-effectiveness of HIV testing for rural trauma patients is questionable with incidences between 0.5 and 1.0 per cent.

Experiential constraints on the development of tolerance to amphetamine hypophagia following sensitization of stereotypy: instrumental contingencies regulate the expression of sensitization.

In previous research, sensitization of stereotypy induced by injections of 2.5 mg/kg amphetamine did not interfere with subsequent tolerance development to the hypophagic effect of 2 mg/kg. This study examined the effect of a higher sensitizing dose. Rats given intermittent injections of 5 mg/kg amphetamine and then challenged with various doses of amphetamine showed focused head scanning at 2 mg/kg and oral stereotypy at 4 mg/kg. In contrast, saline controls showed diffuse sniffing and head scanning at 2 and 4 mg/kg. Subgroups from each condition were then given daily injections of either amphetamine (2 mg/kg) or saline and access to milk for 30 min. Dose-response tests revealed that both drugged groups learned to suppress stereotypy in order to feed at 2 mg/kg, but only the non-sensitized group could do so at 4 mg/kg. These results demonstrate that (1) rats learn to suppress only those stereotyped movements that they experience in the context of feeding and (2) instrumental contingencies can influence the expression of behavioral sensitization.

Role of behavioral and pharmacological variables in the loss of tolerance to amphetamine hypophagia.

Tolerance to amphetamine-induced hypophagia is lost when drug injections are withdrawn for 4 weeks while milk tests are continued (Wolgin and Hughes 1996). The purpose of this study was to determine whether the loss of tolerance is a function of drug withdrawal per se. Rats made tolerant to amphetamine (2 mg/kg, IP) were assigned to one of three groups. During the next 4 weeks (phase), one group continued to receive amphetamine injections prior to daily milk tests (Before group), one group received drug injections after the milk tests (After group), and one group received injections of saline prior to the milk tests (Saline group). Dose-response tests revealed that the Before group retained tolerance, whereas the After and Saline groups lost tolerance. When retested with chronic injections of amphetamine prior to milk, the After and Saline groups reacquired tolerance more rapidly, and to a greater extent, than non-tolerant controls. These results demonstrate that the loss of tolerance is not due to drug withdrawal per se, but may be due to the unlearning of behavioral strategies previously acquired under the drug.

Complications of epidural infusions for analgesia in postoperative and trauma patients.

Few studies compare complications of continuous and bolus epidural analgesia. Ninety-eight postoperative and trauma patients receiving epidural infusions over 15 months were retrospectively studied. Continuous epidural analgesia was used for pain management in 60 patients (61%). Bolus epidural analgesia was administered to 38 patients (39%). Sixty patients reported 98 complications. Sixty-eight per cent of complications occurred in patients receiving continuous infusions. For the continuous infusions, motor blockade (18%), nausea/vomiting, (18%), and catheter leaks (12%) were the most common complications. For bolus infusions, nausea/vomiting (25%), mental status changes (21%), and erythema at placement site (13%) were encountered. Continuous infusions were associated with an increased incidence of complications compared with bolus infusions (P < 0.05). Patients undergoing abdominal surgery had an increased incidence of complications compared with other patients (P < 0.05). Epidural catheters are safe and effective for pain management, but they are not without risk. Hemodynamic stability and pulmonary status should be considered when evaluating patients.

Effect of sensitization of stereotypy on the acquisition and retention of tolerance to amphetamine hypophagia.

The purpose of this study was to determine whether prior sensitization of stereotypy interferes with the development and retention of tolerance to amphetamine-induced hypophagia. Rats were given intermittent injections of either amphetamine (2.5 mg/kg) to induce sensitization of stereotypy, or saline. Subgroups from each group then received daily injections of either amphetamine (2 mg/kg) or saline and access to milk for 30 min. Both sensitized and nonsensitized groups became tolerant to drug-induced hypophagia at about the same rate and to about the same extent. Such tolerance was accompanied by a decrease in the frequency of stereotyped movements while milk was available. The rats were then given daily milk tests for 4 weeks without injections. Subsequent tests with amphetamine revealed that both groups lost tolerance to drug-induced hypophagia and displayed more intense stereotypy than they had prior to drug withdrawal. We conclude that sensitization of stereotypy produced by intermittent injections of amphetamine (2.5 mg/kg) does not retard the development of tolerance to drug-induced hypophagia and does not alter the rat's ability to suppress stereotyped movements. However, the loss of tolerance following drug withdrawal may have been due to the development of more intense stereotypy and/or the "unlearning" of previously acquired strategies for suppressing stereotypy.

Estrogen receptor expression in human pituitary: correlation with immunohistochemistry in normal tissue, and immunohistochemistry and morphology in macroadenomas.

Forty-one human pituitary adenoma specimens were examined for the presence of estrogen receptor (ER) messenger ribonucleic acid and protein using a combination of ribonuclease protection assay, [3H] estradiol ([3H]E2) binding, and ER immunohistochemistry. ER messenger ribonucleic acid prevalence was high in PRL-immunoreactive tumors (2 of 2), moderate in GH/PRL tumors (2 of 5), and low or absent (0 of 4) in GH tumors. In the GH/PRL-immunostaining tumors, the presence of the ER was uniformly associated with elevated serum PRL levels. Among the gonadotropin-immunostaining tumors, 10 of 17 were ER positive; within this group, those with gonadotroph adenoma characteristics were ER positive, whereas those with null cell/oncocytic characteristics were ER negative. Of the tumors that did not immunostain for any known anterior pituitary hormones, 3 of 11 were ER positive. ER immunohistochemistry in 14 tumors revealed a 100% correlation with ribonuclease protection assay results, whereas [3H]E2 binding, determined in 9 tumors, showed an 87% correlation. In summary, it appears that PRL and a specific class of gonadotropin-immunostaining tumors (identifiable by specific characteristics on electron microscope) contain ER, whereas GH-immunostaining tumors are ER negative. ER expression in normal pituitary paralleled that in macroadenomas (GH, 2.3%; PRL, 50%; FSH, 70%; LH, 83%; TSH, 4%; ACTH, 1%). The ER-positive tumors represent a subset whose growth and secretory profiles may be influenced by the gonadal steroidal milieu or by pharmacological agents that affect E2 levels or ER function.

Measurement of oxybutynin and its N-desethyl metabolite in plasma, and its application to pharmacokinetic studies in young, elderly and frail elderly volunteers.

1. A quantitative h.p.l.c. plasma assay for oxybutynin (OB) and its active metabolite, N-desethyl oxybutynin (DEOB) is described. The method is linear with coefficients of variation ranging between 4 and 11.8% for OB and 4.6-9.1% for DEOB over the typical concentration range measured. Minimum detectable levels were 0.5 and 5 ng/ml for OB and DEOB respectively from a 2 ml sample. 2. Pharmacokinetic parameters were obtained after a single oral dose of OB and after administration two or three times daily to frail elderly and elderly volunteer groups respectively. Single dose results were also compared with data from young healthy volunteers. 3. There was a wide range in peak blood levels and high levels of parent drug were matched by high DEOB metabolite levels. Plasma levels on repeated administration were as would be predicted from the single dose kinetics. 4. Area under the plasma time course curve for DEOB metabolite was less than or equal to 5 than that of the parent drug. 5. A trend of increasing peak plasma levels and bioavailability was observed with increasing age and frailty, with the differences more apparent between the active elderly and frail elderly groups than between the active elderly and young volunteers. 6. Results indicate that for frail elderly patients a lower initial starting dose of 2.5 mg OB given two or three times a day may provide adequate therapeutic blood levels of the drug.