Production of a baculovirus-derived gp50 protein and utilization in a competitive enzyme-linked immunosorbent assay for the serodiagnosis of pseudorabies virus

The simultaneous detection of the green fluorescent protein (GFP) and DNA content using propidium iodide (PI) by flow cytometry is made difficult because of the unique nature of these 2 fluorogenic reagents. For PI to enter cells efficiently and to stain DNA quantitatively, the cells must first be permeabilized; ethanol treatment is a routine method to achieve this. However, this permeabilization step causes GFP, which is normally found in the cytoplasm, to leach out of the cells. Although the use of paraformaldehyde-based fixatives allows GFP to be maintained in cells and retain its fluorescence even after ethanol permeabilization, the protocol we commonly employ results in inefficient PI staining and poor quality DNA histograms. To circumvent these difficulties, we have employed a GFP-fusion protein which localizes to the cellular membrane and as such is retained in cells upon ethanol permeabilization without prior fixation. This allows the GFP signal to be detected in cells treated with ethanol in preparation for PI staining and cell cycle analysis. This property facilitates the use of GFP as a marker for transfected cells in experiments designed to characterize the effects of ectopic expression of cellular or viral genes on cell cycle progression.     

A new biochemical assay in the diagnostic management of nasal cerebrospinal fluid leakage

A new method for the detection of cerebrospinal fluid (CSF) leakage is described, and is a refinement of the method originally reported by Oberascher and Arrer in 1986. Immuno-electrophoretic measurements are performed in a two-buffer system, making the test easier to do and providing qualitatively better images. Genetic variants of transferrin (which has a general population incidence of 2-4%) can be discriminated from false-positive test results in affected families. The test described is recommended as the method of choice for initial screening of suspected CSF leakage.     

Comparison of use of cerebrospinal fluid, serum, and throat swab specimens in diagnosis of enteroviral acute neurological infection by a rapid RNA detection PCR assay

A PCR assay for detection of enterovirus RNA in multiple specimen types from patients with neurological infections was evaluated. Combined PCR assay of cerebrospinal fluid and serum (systemic specimens) was more sensitive than assaying either specimen alone in children but not in adults. Compared with PCR in systemic specimens, detection of enterovirus RNA in throat swabs showed a sensitivity of 62.5% and a specificity of 75.6%.     

A competitive enzyme-linked immunosorbent assay for measuring the levels of serum antibody to Haemophilus influenzae type b

1. The protein family of the neurotrophins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and Neurotrophin-3, -4/5, and -6 (NT-3; NT-4/5; NT-6) is well known to enhance the survival and to stabilize the phenotype of different populations of neurons in the central and the peripheral nervous system. These effects are mediated via binding to specific tyrosine kinase receptors (Trks) and to the low-affinity p75 neurotrophin receptor. 2. The neurotrophins NGF, BDNF, and NT-3 and the BDNF and NT-3 selective receptors (TrkB, TrkC) are expressed at high levels in neurons of the basal forebrain, the hippocampus, and the neocortex of the mammalian brain. The expression and secretion of NGF and BDNF in these brain areas is regulated by (physiological levels of) neuronal activity. 3. Exogenous application of the neurotrophins to hippocampal and neocortical neurons can enhance excitatory glutamatergic synaptic transmission via activation of Trk receptors. In addition, long-term potentiation (a potential cellular correlate for learning and memory formation in mammals) in the rodent hippocampus depends on endogenous supply of neurons with BDNF. 4. Judged by the analysis of electrophysiological data, the BDNF- and NT-3-induced enhancement of glutamatergic synapses is mediated by increasing the efficacy of glutamate release from the presynaptic neuron. However, neurotrophin-dependent postsynaptic enhancement of NMDA (but not AMPA) receptor responsiveness has also been shown. 5. On the molecular level, neither the pre- nor the postsynaptic modulation of glutamatergic synapses by neurotrophins is well understood. However, neurotrophins were shown to acutely affect intraneuronal Ca2+ levels and to influence molecular components of the transmitter release machinery, which could underly the presynaptic modifications, whereas BDNF-induced phosphorylation of NMDA-type glutamate receptors could account for the postsynaptic effects. 6. Taken together, these results suggest that the activity-dependent release of neurotrophins at frequently used synapses could modulate the synaptic efficacy at these junctions. Thus, neurotrophins might operate as locally released feedback modulators of synaptic transmission, and this could be a cellular correlate for certain aspects of information processing in the mammalian brain.     

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fluid

A polymerase chain reaction (PCR) method for the rapid diagnosis of tuberculous meningitis (TBM) was used to study prospectively 47 cerebrospinal fluid (CSF) samples from 45 patients. Twenty CSF samples were from patients with clinically suspected TBM and another 27 samples came from patients without clinically suspected TBM. Mycobacterial DNA was detected in 15 CSF samples (14 from patients with clinically suspected TBM and 1 from a patient not suspected of having TBM). Of the PCR-positive samples, 4 were also positive for mycobacterial culture. However, 32 PCR-negative samples were all culture-negative. All samples were negative for the acid-fast bacillus by direct smear. The single PCR-positive patient in the clinically unsuspected TBM group was initially diagnosed as suffering from aseptic meningitis on the basis of his clinical features. The mycobacterial culture of his CSF specimen was also positive and a revised diagnosis of an aseptic type of TBM was made. The estimations of specificity and sensitivity in this study were 100% and 70% respectively. The results showed that using a PCR to detect mycobacterial DNA in CSF for the early diagnosis of TBM is not only a rapid but also an accurate method.     

Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive polymerase chain reaction and enzyme-linked immunosorbent assay

The rising interest in gene therapy for the treatment of numerous disorders necessitates the need for the large-scale production of therapeutic biopharmaceuticals that meet stringent purity standards. Residual host cell DNA in recombinant pharmaceuticals has been identified as a potential risk factor that must be quantitated carefully both during the manufacturing process and in the final product. We describe a PCR method to quantitate contaminating levels of host cell DNA in clinical plasmid DNA preparations intended for human gene therapy. The quantitation is based on the coamplification of two similar templates, the target DNA and a synthetic competitor, and the quantitation of the resulting PCR products. The competitor is identical to the target DNA PCR product except for a 29-bp internal replacement. As a result, the two PCR products can easily be distinguished from each other. The competitive nature of the assay allows the use of the ratio of the target DNA PCR product to the competitor DNA PCR product to determine the original amount of target DNA in a sample. The primers used in this assay anneal to a conserved region of the E. coli 23S rRNA gene. One of the primers is biotinylated, allowing the PCR products to be detected colorimetrically after their capture on microtiter plates. The capture is accomplished by differential hybridization to target and competitor-specific probes covalently attached to wells of microtiter plates. The entire assay is performed in less than 2 hr postamplification. This method represents an attractive alternative to Southern blot analysis, which is the currently established method for DNA quantitation.     

Amphetamines in hair by enzyme-linked immunosorbent assay

Human hair was collected from the occipital crown region of the head from several subjects; these hair samples were presumptively positive for amphetamines by a previously evaluated immunoassay. Hair was washed briefly with methanol to remove external contamination, then extracted with hot methanol for 2 h to recover the drugs. The extracts were evaporated to dryness, reconstituted in buffer, and analyzed using a new enzyme-linked immunosorbent assay (ELISA) technique adapted for the detection of amphetamines in hair. Gas chromatography-mass spectrometry was used as the reference technique. Cross-reactivity of several related compounds was evaluated by equating the inverse of the ligand concentration at 50% antibody binding to the affinity constant for each compound. The ratio of a compound's affinity constant to that for d-methamphetamine was used to derive percent crossreactivity. These experiments yielded values of 30.8% for d-amphetamine, 7.4% for I-methamphetamine, 4.3% for phentermine, 2.9% for I-amphetamine, and <1% for ephedrine, methylenedioxyamphetamine, and methylenedioxymethamphetamine. Cross-reactivity of unrelated compounds was found to be non-existent. The optimum cutoff concentration was determined by receiver operating characteristic curve analysis to be 300 pg/mg and the observed limit of detection was 60 pg/mg. Intra-assay precision at 300 pg/mg was 3.3% (coefficient of variation, CV), and the interassay CV was 10.5%. The sensitivity and specificity of the method were 83% and 92%, respectively.     

Immunoaffinity column as cleanup tool for a direct competitive enzyme-linked immunosorbent assay of cyclopiazonic acid in corn, peanuts, and mixed feed

PURPOSE: Atherothrombotic occlusion of the cervical internal carotid artery (ICA) without collateral flow is one of the most critical forms of acute ischemia. We report the results of urgent thrombolytic treatment of patients with major stroke in this clinical category. METHODS: Clinical findings and outcome in 33 patients were investigated. All patients had suffered a major stroke, with a score of 24 or higher on the NIH Stroke Scale on admission. Ischemic abnormalities were not detected on initial CT studies. Diagnoses were made at angiography, and patients were treated by intravenous or intraarterial local thrombolysis within 6 hours of stroke onset. RESULTS: Recanalization was accomplished in eight patients with intraarterial local thrombolysis; four of these patients had a good clinical outcome. Two factors characteristic of those whose treatment was successful were dramatic improvement of symptoms after partial recanalization achieved within 3 hours of onset and stabilized improvement after subsequent percutaneous transluminal angioplasty or carotid endarterectomy for residual atherosclerotic stenosis at the ICA origin. CONCLUSION: The results of this study suggest that urgent intraarterial local thrombolysis may be a successful treatment method for some patients in this critical clinical category if the treatment can be accomplished within 3 hours of ictus and followed by either angioplasty or endarterectomy for residual stenosis.     

Antibiotic pharmacodynamics in cerebrospinal fluid

The CSF half-lives of lipophilic agents, such as quinolones, are similar to those in serum and peak concentrations in CSF are achieved relatively quickly. In contrast, the pharmacokinetics of hydrophilic agents (beta-lactams and vancomycin) in CSF often differ from those in serum. In particular, the half-lives of these agents in CSF tend to be extended, and the time to achieve peak concentrations in CSF is delayed. Hydrophilic antibiotics, such as beta-lactams, penetrate poorly through the BBB, but CSF penetration is significantly increased in the presence of inflammation. In contrast, lipophilic antibiotics, such as quinolones, enter the CSF more efficiently and their penetration is not inflammation dependent. The pharmacodynamic properties of antibiotics in CSF are generally similar to those in other body sites; beta-lactam agents and vancomycin are time-dependent, whereas the quinolones and aminoglycosides are concentration-dependent. However, a notable difference from infections in other sites is that quinolones have a short PAE in CSF and need to continually exceed the MBC for maximal effectiveness. Thus, in CSF, quinolones demonstrate features of both concentration-dependency and time-dependency, evidence that the AUC/MBC is an important predictor of effectiveness. With the exception of quinolones, many antibiotics appear to have prolonged sub-MIC effects and longer half-lives in CSF than in serum, suggesting that dosing intervals longer than those used traditionally would be effective in meningitis. However, this requires clinical verification.     

Hyaluronan in human cerebrospinal fluid

We studied the concentration of hyaluronan in cerebrospinal fluid (CSF) in various diseases and attempted to define its reference interval. A radioassay utilizing cartilage proteins with affinity for hyaluronan was used in determining the concentration of 200 lumbar and 27 ventricular CSF specimens and 11 brain cyst fluids. Molecular weight distributions were determined by gel chromatography and localization in brain tissue by histochemistry. The hyaluronan level of lumbar CSF showed an increase with age; comparatively healthy children had (mean +/- SD) 50 +/- 41 micrograms/L (n = 40) and adults 166 +/- 77 micrograms/L (n = 9); i.e. significantly different values. The highest level was recorded in a patient with meningitis (> 8000 micrograms/L). More than 4000 micrograms/ L was noted in a patient with tumour metastasis in the cerebellum. Significantly elevated levels were especially found with spinal stenosis, head injury and cerebral infarction, but also in inflammatory medical disorders, hydrocephalus and encephalitis. We found no significant increase in multiple sclerosis and some other neurological diseases. Ventricular CSF of adults contained significantly less hyaluronan (53 +/- 73 micrograms/L; n = 16) than lumbar CSF. Hyaluronan in cyst fluids varied from 31 to 25,000 micrograms/L. Weight average molecular weight of hyaluronan in CSF was 2.9-3.0 x 10(5) and in brain tumour cyst fluid 2.4 x 10(6). In search for the origin of hyaluronan in CSF it was found that its concentration in the choroid plexus and leptomeninges was low, but that hyaluronan was accumulated in the superficial layer of the cerebral cortex. Continued screening for hyaluronan in CSF may be valuable in cases of inflammatory diseases, tumours and obstruction to CSF flow.     

Beta-trace protein in cerebrospinal fluid: a blood-CSF barrier-related evaluation in neurological diseases

Beta-trace protein concentrations in cerebrospinal fluid (CSF) and serum from 113 patients with various neurological diseases and 65 controls were determined with a sensitive and specific immunonephelometric assay. In adult control patients, beta-trace concentrations were 14.6+/-4.6 mg/L in CSF and 0.46+/-0.13 mg/L in serum, that is, 32-fold higher in CSF. beta-trace levels in CSF correlated with age as well as with the albumin CSF/serum ratio (Q(Alb)), which is considered a measure for blood-CSF barrier function. The relationship between CSF beta-trace levels and elevated Q(Alb) values was studied in various neurological diseases with CSF protein increase. In spinal canal stenosis, CSF beta-trace (mean=29.5+/-10.5 mg/L) correlated positively with increasing Q(Alb) values. In bacterial meningitis, CSF beta-trace (mean=8.7+/-3.9 mg/L) remained invariant to changes of Q(Alb) values. In Guillain-Barré syndrome, CSF beta-trace (mean=14.4+/-6.8 mg/L) was below the Q(Alb)-dependent reference range. In multiple sclerosis and viral meningoencephalitis, beta-trace levels were within the reference range. Beta-trace concentration in CSF can be used in conjunction with Q(AlB) to distinguish between different neurological pathologies associated with CSF protein increase.     

A high-performance liquid-chromatographic microanalytical procedure for the rapid estimation of piracetam in plasma or cerebrospinal fluid

Presently available GC and HPLC methods for analysis of piracetam, require large samples and suffer from interference. A micro scale, isocratic high-performance liquid-chromatographic method is described for the determination of piracetam in plasma (25 microL) or cerebrospinal fluid (10 microL) using ultraviolet absorbance at 215 nm. The limit of quantitation is 4 micrograms mL-1 and the within-batch and between-batch coefficients of variation are less than 10%. No interference from other commonly prescribed antimyoclonic or antiepileptic drugs was observed and thus the method can be used to monitor piracetam in patients on polytherapy antimyoclonic or antiepileptic drug regimens. Because of the sensitivity and rapidity of the method it is suitable for pharmacokinetic and mechanistic studies and for analysis of paediatric samples.     

Nephritis associated with a diphtheroid-infected cerebrospinal fluid shunt

Hypocomplementemic proliferative glomerulonephritis occurred during diphtheroid infection of a ventricular decompression shunt for cerebrospinal fluid diversion (cerebrospinal fluid shunt) in a young man. Granular deposits of immunoglobulin M (IgM) and the third component of complement (C3) were found along the glomerular basement membrane. This report provides supportive evidence for immune complex-mediated glomerular injury due to diphtheroid infection in a cerebrospinal fluid shunt.     

Detection of flunixin in greyhound urine by a kinetic enzyme-linked immunosorbent assay

Anxiety levels in a sample of 65 long-term cancer survivors were assessed in a study of the effects of a planned discharge from an oncology clinic. Thirty-one percent of patients scored > or = 8, and 12% > or = 11 on the anxiety subscale of the Hospital Anxiety and Depression Scale (HADS), indicating that anxiety rates in patients in long-standing remission do not greatly differ from patients with active disease. Despite the provision of continued support and guaranteed fast-access return to the clinic if necessary, 28% of patients refused to be discharged. Fear that recurrence would not be detected was the reason most frequently cited. Seventy-five percent of these patients were HADS anxiety cases. A second assessment 4-5 months later of the 41 patients who were discharged showed a slight, but non-significant increase in anxiety rates suggesting that anxiety in cancer survivors may be persistent and not related to clinic attendance.     

Enlarged cerebrospinal fluid spaces in opiate-dependent male patients: a stereological CT study

Total knee arthroplasty was evaluated in 10 patients with post-traumatic osteoarthrosis secondary to work-related knee injuries (age- and sex-matched with 10 controls who had total knee arthroplasties for nonwork-related osteoarthrosis) to determine if Workers' Compensation status influenced treatment outcome. Using the Hospital for Special Surgery Knee Rating System (maximum possible score: 100), most recent follow-up scores averaged 64.1 for Workers' Compensation patients and 91.9 for controls. Subjective indices (pain, function) were significantly different between groups (p < 0.05), but objective indices (range of motion, strength, deformity, instability) were not. No significant differences were noted between groups on either immediate postoperative or most recent follow-up radiographs (which were assessed for alignment and radiolucencies at implant surfaces, respectively). Suboptimal outcomes can be anticipated in total knee arthroplasties performed on Workers' Compensation patients, particularly in cases where claims have not been settled at the time of surgery.     

Assay of gentamicin in cerebrospinal fluid

A comparison of standard curves obtained from a conventional plate diffusion assay method revealed significant differences when gentamicin standards were made up in different media. Standards made up in distilled water resulted in a curve which differed from that of standards made up in pooled human cerebrospinal fluid by a factor of up to 4. When the assay medium was supplemented with 0-5% sodium chloride, the difference between the two standard curves was reduced to a factor of about 1-5. The curve obtained from standards made up in 150 mM sodium chloride/4-5 mM calcium chloride correlated well with that from standards made up in cerebrospinal fluid. There was no evidence of gentamicin being bound to protein in the cerebrospinal fluid.     

Coccidioides immitis presenting as a hyphal form in cerebrospinal fluid

This article reports a case of Coccidioides immitis that presented as a hyphal form in a 38-year-old patient. The organism was observed growing exclusively as hyphae in the cerebrospinal fluid by microscopic examination. Coccidioides immitis was the only organism cultured. The identification of C immitis was confirmed by both standard culture methods and DNA probe studies.     

Amniotic fluid lipid profiles: a rapid gas chromatographic method

The lipid extract of amniotic fluid has been analysed for the important fatty acids derived mainly from the lecithin component of lung surfactant. Using gas-liquid chromatography and mass spectrometry, these fatty acids have been identified. A positive correlation between certain lipid profiles and lack of lung surfactant with its associated respiratory problems for the newborn infant has been demonstrated.     

Evaluation of a commercially available reverse transcription-PCR assay for diagnosis of enteroviral infection in archival and prospectively collected cerebrospinal fluid specimens

BACKGROUND: In the Fontan circulation, energy consumption at the cavopulmonary connection is crucial. Our hypothesis was that a modification of the standard Norwood variant of cavopulmonary connection with an extended anastomosis would improve hydrodynamics. METHODS: The in vitro hydrodynamics of two different Perspex glass models resembling the Norwood variant of cavopulmonary connection (model I) and the modification (model II) were analyzed in a mock circulation at nonpulsatile flows of 2 to 5 L/min to simulate rest and exercise. The pulmonary flow split was varied to imitate varying lung resistances. Inferior-to-superior caval flow ratio and size of models were increased to simulate growth. RESULTS: The pulmonary flow was preferentially directed to the left lung in model I and was better balanced in model II. Power losses increased exponentially with total flow in both models and were markedly higher in model I. These differences were attenuated in the larger models. Anastomotic turbulences were larger in model I. Power losses in both models were relatively insensitive to changes in pulmonary flow split. CONCLUSIONS: The proposed modification of the Norwood variant of cavopulmonary connection seems to be hydrodynamically advantageous and warrants further evaluation.     

In vivo osteogenesis assay: a rapid method for quantitative analysis

A quantitative in vivo osteogenesis assay is a useful tool for the analysis of cells and bioactive factors that affect the amount or rate of bone formation. There are currently two assays in general use for the in vivo assessment of osteogenesis by isolated cells: diffusion chambers and porous calcium phosphate ceramics. Due to the relative ease of specimen preparation and reproducibility of results, the porous ceramic assay was chosen for the development of a rapid method for quantitating in vivo bone formation. The ceramic cube implantation technique consists of combining osteogenic cells with 27-mm3 porous calcium phosphate ceramics, implanting the cell-ceramic composites subcutaneously into an immuno-tolerant host, and, after 2-6 weeks, harvesting and preparing the ceramic implants for histologic analysis. A drawback to the analysis of bone formation within these porous ceramics is that the entire cube must be examined to find small foci of bone present in some samples; a single cross-sectional area is not representative. For this reason, image analysis of serial sections from ceramics is often prohibitively time-consuming. Two alternative scoring methodologies were tested and compared to bone volume measurements obtained by image analysis. The two subjective scoring methods were: (1) Bone Scale: the amount of bone within pores of the ceramic implant is estimated on a scale of 0-4 based on the degree of bone fill (0=no bone, 1=up to 25%, 2=25 to 75%, 4=75 to 100% fill); and (2) Percentage Bone: the amount of bone is estimated by determining the percentage of ceramic pores which contain bone. Every tenth section of serially sectioned cubes was scored by each of these methods under double-blind conditions, and the Bone Scale and Percentage Bone results were directly compared to image analysis measurements from identical samples. Correlation coefficients indicate that the Percentage Bone method was more accurate than the Bone Scale scoring method. The Bone Scale scoring method gave an r2=0.767 while the Percentage Bone method gave a value of 0.902. These results indicate that scoring ceramic cubes by the percentage of pores containing bone gives a result that corresponds to image analysis measurements at nearly a 90% confidence level. Thus, the Percentage Bone method of scoring is an accurate and relatively quick scoring method for in vivo bone formation.