Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results.

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.

Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

Perampanel: a significant liver enzyme inducer in some patients?

Perampanel is one of the latest released antiepileptic drugs (AEDs). Early studies suggest no significant liver enzyme induction from this compound. We report on two patients with medically resistant epilepsy, who had perampanel added to their usual regimen. Both experienced a worsening of their epilepsy and presented in convulsive status epilepticus; concurrent antiepileptic drug levels (phenytoin, phenobarbital, rufinamide) were significantly decreased (<50%) in comparison with levels prior to perampanel introduction. Intravenous load and significant increase of maintenance dosages were needed to restore therapeutic drug levels. In one patient, further increase of perampanel resulted in a new drop of phenytoin level. This suggests that perampanel could, in some subjects, induce liver enzymes and interact with concomitant AEDs; monitoring levels of concomitant compounds could be useful.

Liver enzyme elevation after lamivudine withdrawal in HIV-hepatitis B virus co-infected patients: the Swiss HIV Cohort Study.

Background The principal causes of liver enzyme elevation among HIV-hepatitis B virus (HBV) co-infected patients are the hepatotoxic effects of antiretroviral therapy (ART), alcohol abuse, ART-induced immune reconstitution and the exacerbation of chronic HBV infection. Objectives To investigate the incidence and severity of liver enzyme elevation, liver failure and death following lamivudine (3TC) withdrawal in HIV-HBV co-infected patients. Methods Retrospective analysis of the Swiss HIV Cohort Study database to assess the clinical and biological consequences of the discontinuation of 3TC. Variables considered for analysis included liver enzyme, HIV virological and immunological parameters, and medication prescribed during a 6-month period following 3TC withdrawal. Results 3TC was discontinued in 255 patients on 363 occasions. On 147 occasions (109 patients), a follow-up visit within 6 months following 3TC withdrawal was recorded. Among these patients, liver enzyme elevation occurred on 42 occasions (29%), three of them (2%) with severity grade III and five of them (3.4%) with severity grade IV elevations (as defined by the AIDS Clinical Trials Group). Three patients presented with fulminant hepatitis. One death (0.7%) was recorded. Conclusions HBV reactivation leading to liver dysfunction may be an under-reported consequence of 3TC withdrawal in HIV-HBV co-infected patients. Regular monitoring of HBV markers is warranted if active therapy against HBV is discontinued.

Effect on blood pressure and the renin-angiotensin system of repeated doses of the converting enzyme inhibitor CGS 14824A.

A new, orally active angiotensin converting enzyme (ACE) inhibitor, CGS 14824A, was evaluated in 12 healthy male volunteers. Two groups each of 6 volunteers were given 5 or 10 mg once daily p.o. for 8 days. Four hours after the first and the last morning doses, plasma angiotensin II, aldosterone and plasma converting enzyme activity had fallen, while blood angiotensin I and plasma renin activity had risen. Throughout the study, more than 90% inhibition of ACE was found immediately before giving either the 5 or 10 mg dose and 50% blockade was still present 72 h following the last dose. Based on the determination of ACE, there was no evidence of drug accumulation. No significant change in blood pressure or heart rate was observed during the course of the study. CGS 14824A was an effective, orally active, long-lasting and well tolerated converting enzyme inhibitor.

Beyond intracranial pressure: optimization of cerebral blood flow, oxygen, and substrate delivery after traumatic brain injury.

Monitoring and management of intracranial pressure (ICP) and cerebral perfusion pressure (CPP) is a standard of care after traumatic brain injury (TBI). However, the pathophysiology of so-called secondary brain injury, i.e., the cascade of potentially deleterious events that occur in the early phase following initial cerebral insult-after TBI, is complex, involving a subtle interplay between cerebral blood flow (CBF), oxygen delivery and utilization, and supply of main cerebral energy substrates (glucose) to the injured brain. Regulation of this interplay depends on the type of injury and may vary individually and over time. In this setting, patient management can be a challenging task, where standard ICP/CPP monitoring may become insufficient to prevent secondary brain injury. Growing clinical evidence demonstrates that so-called multimodal brain monitoring, including brain tissue oxygen (PbtO2), cerebral microdialysis and transcranial Doppler among others, might help to optimize CBF and the delivery of oxygen/energy substrate at the bedside, thereby improving the management of secondary brain injury. Looking beyond ICP and CPP, and applying a multimodal therapeutic approach for the optimization of CBF, oxygen delivery, and brain energy supply may eventually improve overall care of patients with head injury. This review summarizes some of the important pathophysiological determinants of secondary cerebral damage after TBI and discusses novel approaches to optimize CBF and provide adequate oxygen and energy supply to the injured brain using multimodal brain monitoring.

Individual responses to converting enzyme inhibitors and calcium antagonists.

This study was designed to assess whether the acute blood pressure response of an individual hypertensive patient to a calcium antagonist or an angiotensin converting enzyme (ACE) inhibitor is a good predictor of the long-term efficacy of these drug classes in this particular patient. The concept that good responses to ACE inhibitors and calcium antagonists may be mutually exclusive was also tested. Sixteen patients were included in a randomized crossover trial of enalapril, 20 mg daily, and diltiazem, 120 mg daily, for 6 weeks each. Blood pressure was measured by ambulatory blood pressure recording. During the washout phase, the acute effect of nifedipine, 10 mg p.o., and enalaprilat, 5 mg i.v., was evaluated. Nifedipine and enalaprilat reduced blood pressure equally well. The long-term blood pressure reduction induced by enalapril and diltiazem was similar. The acute blood pressure response to a given drug was not a good predictor of the result obtained with long-term therapy. No age dependency of the antihypertensive effect of either drug class was apparent. There was no evidence that a good response to one drug excluded a similarly good response to the other.

Effects of prolonged administration of the angiotensin converting enzyme inhibitor CGS 16617 in normotensive volunteers.

A new, orally active angiotensin converting enzyme (ACE) inhibitor, CGS 16617, has been evaluated in normotensive subjects during acute and prolonged administration. Single ascending doses of CGS 16617 20 to 100 mg were given to 9 normotensive volunteers at one week intervals and the changes in blood pressure, plasma ACE and renin activity were examined up to 72 h after drug intake. Also, CGS 16617 50 mg/day or placebo were given for 30 days to 8 and 6 normotensive subjects, respectively, maintained on an unrestricted salt diet. Blood pressure was measured daily in the office and ambulatory blood pressure profiles were also obtained before, during and after therapy, using the Remler M 2000 blood pressure recording system. CGS 16617 was an effective and long lasting ACE inhibitor. It did not induce a consistent change in blood pressure, but, the individual responses were very variable and several subjects experienced a clear decrease in the average of the blood pressures recorded during the daytime.

Renal hemodynamic and natriuretic effects of concomitant Angiotensin-converting enzyme and neutral endopeptidase inhibition in men.

This double-blind placebo-controlled study was designed to investigate the acute and sustained hormonal, renal hemodynamic, and tubular effects of concomitant ACE and neutral endopeptidase (NEP) inhibition by omapatrilat, a vasopeptidase inhibitor, in men. Thirty-two normotensive subjects were randomized to receive a placebo, omapatrilat (40 or 80 mg), or the fosinopril/hydrochlorothiazide (FOS/HCTZ; 20 and 12.5 mg, respectively) fixed combination for 1 week. Blood pressure, renal hemodynamics, urinary electrolytes and atrial natriuretic peptide excretion, and several components of the renin-angiotensin system were measured for 6 hours on days 1 and 7 of drug administration. When compared with the placebo and the FOS/HCTZ combination, omapatrilat induced a significant decrease in plasma angiotensin II levels (P<0.001 versus placebo; P<0.05 versus FOS/HCTZ) and an increase in urinary atrial natriuretic peptide excretion (P<0.01). These hormonal effects were associated with a significant fall in blood pressure (P<0.01) and a marked renal vasodilatation, but with no significant changes in glomerular filtration rate. The FOS/HCTZ markedly increased urinary sodium excretion (P<0.001). The acute natriuretic response to FOS/HCTZ was significantly greater than that observed with omapatrilat (P<0.01). Over 1 week, however, the cumulative sodium excretion induced by both doses of omapatrilat (P<0.01 versus placebo) was at least as great as that induced by the dose of FOS/HCTZ (P=NS versus FOS/HCTZ). In conclusion, the results of the present study in normal subjects demonstrate that omapatrilat has favorable renal hemodynamic effects. Omapatrilat combines potent ACE inhibition with a sustained natriuresis, which explains its well-documented potent antihypertensive efficacy.

Energy substrate used by workers of leaf-cutting ants during nest excavation

Energy substrate used by workers of leaf-cutting ants during nest excavation. In this study we aimed to ascertain whether leaf-cutting ant workers lose body reserves (fat or sugars) as a function of nest excavation. For each treatment, we isolated 10 workers of Atta sexdens into two experimental groups, Control (C- without excavation) and Soil (S- with excavation), which were kept for different time intervals (0, 24, 48 or 72 hours), totaling 700 tested workers. We then determined the concentration of soluble carbohydrates and total lipid content in them. The total carbohydrates were determined colorimetrically, based on the reaction between carbohydrates and sulfuric acid-phenol. For determination of lipids, the insects were immersed in organic solvent until they reached a constant weight. Our results showed that carbohydrates are consumed during nest excavation activities. In the experimental groups S24, S48 and S72, there was an average reduction of 5.82 (20.42%), 14.31 (44.96%) and 13.27 (43.96%) µ.mg-1 in soluble sugar when compared with the experimental groups that did not excavate. Furthermore, the lipids were not used during this activity. With respect to dry mass of the workers, their values were C0 = 8%, C24 = 10.4%, C48 = 9.2%, C72 = 10%, S24 = 9.2%, S48 = 8.7% and S72 = 8.5%. Our results show experimentally that the source of energy for nest excavation is carbohydrates, whereas lipids are conserved for other activities.

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

Characterization of the enzyme involved in the processing of big endothelin-1 in human lung epithelial cells.

Biosynthesis of active endothelin-1 (ET-1) implies an enzymatic processing of the inactive precursor Big ET-1 (1-39) into the mature, 21 amino acid peptide. The aim of this study was to characterize in airway and alveolar epithelial cells the enzymes responsible for this activation. BEAS-2B and A549 cells, which both produce ET-1, were studied in vitro as models for bronchiolar and alveolar cells, respectively. Both cell lines were able to convert exogenously added Big ET-1 (0.1 microM) into ET-1, suggesting a cell surface or an extracellular processing. The conversion was inhibited by phosphoramidon in both cell lines with an IC50 approximately 1 microM, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP). The endogenous production of serum-stimulated BEAS-2B and A549 cells was not inhibited by thiorphan, and phosphoramidon showed inhibition only at high concentration (>100 microM). Western blotting following electrophoresis in reducing conditions demonstrated a protein of MR 110 corresponding to the ECE-1 monomer in both BEAS-2B and A549 cells, as well as in whole lung extracts. By RT-PCR we revealed the mRNA encoding for the ECE-1b and/or -1c subtype, but not ECE-1a, in both cell lines. We conclude that BEAS-2B and A549 cells are able to process either endogenous or exogenous Big ET-1 by ECE-1 and that isoforms 1b and 1c could be involved in this processing with no significant role of NEP.

Development of an enzyme-linked immunosorbent assay for serodiagnosis of ringworm infection in cattle.

The aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms of Trichophyton rubrum dipeptidyl peptidase V (TruDppV) and T. rubrum leucin aminopeptidase 2 (TruLap2), which are 98% identical to Trichophyton verrucosum orthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P < 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.

Role of substrate utilization and thermogenesis on body-weight control with particular reference to alcohol.

Alcohol (ethanol; EtOH) provides fuel energy to the body (29.7 kJ (7. 1 kcal)/g, 23.4 kJ (5.6 kcal)/ml), as do other macronutrients, but no associated essential nutrients. The thermogenic effect of EtOH (on average 15 % of its metabolizable value) is much greater than that of the main substrates utilized by the body, i.e. fat and carbohydrates (CHO), suggesting a lower net efficiency of energy utilization for EtOH than for fat and CHO. EtOH cannot be stored in the body and is toxic, so that there is an obligatory continuous oxidation of EtOH and it becomes the priority fuel to be metabolized. In contrast to CHO, its rate of oxidation does not depend on the dose ingested. As with CHO intake, it engenders a shift in postprandial substrate utilization (decrease in fat oxidation), but by a non-insulin-mediated mechanism. A limited amount of EtOH can be converted to fatty acids by hepatic de novo lipogenesis (as occurs with high levels of CHO feeding) from acetate production, which inhibits lipolysis in peripheral tissues. There is no evidence that EtOH consumed under normoenergetic conditions (i.e. isoenergetically replacing CHO or fat) leads to greater body fat storage than fat or CHO. However, there is still a lack of experimental studies on the influence of EtOH on the level of spontaneous physical activity in man. This effect may well depend on the dose of EtOH consumed as well as other intrinsic factors.