Molecular cloning of agonistic and antagonistic monoclonal antibodies against human 4-1BB.

Previously, we prepared two different monoclonal antibodies (mAbs) against human 4-1BB (CD137): an agonistic mAb BBK-1 and an antagonistic mAb BBK-2. In this paper, we describe the molecular cloning of these two mAbs and present comparisons of their amino acid sequences. cDNAs encoding the heavy (H) and light (L) chains of the two mAbs were cloned by screening of cDNA libraries constructed from hybridomas secreting these mAbs. Comparisons of amino acid sequences of the two mAbs showed that, while the constant regions of the H and L chains were identical between the two mAbs, the variable region showed 45% identity in H chains and 48% identity in L chains. This suggests that these two mAbs recognize different epitopes of 4-1BB and may have different effects on the activity of 4-1BB.     

Comparison of gas-liquid chromatography and EMIT assay for serum valproic acid

We describe a simple direct extraction method for the gas-liquid chromatography determination of serum valproic acid. The working range for the assay is 2-180 mg/L and our within-run precision was 5.8 and 4.3% at the 40 and 90 mg/L concentrations respectively. Hemolyzed and lipemic sera as well as samples from patients with hyperbilirubinemia and from patients with decreased renal function were put through the assay and no interfering peaks were noted. Interference occurred when teflon-lined screw caps were used during the extraction step. The method was proven to be accurate by linear regression analysis of samples containing weighed-in amounts of valproic acid. The above assay was compared to an enzyme immunoassay technique (EMIT). The working range for the latter is 10-150 mg/L and the with-run precision was 10.8 and 5.9% and 90 mg/L concentration respectively. Samples were run by both the gas-liquid chromatograph and enzyme immunoassay methods and gave very similar results over the range 16-139 mg/L.     

Long-term neurotoxicity of chlorpyrifos: spatial learning impairment on repeated acquisition in a water maze.

Organophosphate compounds are cholinesterase inhibitors widely used in agriculture, industry, household products, and even as chemical weapons. Their major mechanism of acute toxic action is the inhibition of acetylcholinesterase, which is responsible for the degradation of the neurotransmitter acetylcholine. An organophosphorus ester-induced chronic neurotoxicity (OPICN) syndrome has been proposed. The OPICN syndrome could result from both long-term exposure to subclinical doses of OPs and after acute poisoning. Development of animal models for the cognitive decline are required and could later help to elucidate the mechanisms involved in this long-term effect on the central nervous system. Previously, we have found performance decrements in a four-trial repeated acquisition spatial task in a water maze. The present study includes two experiments to extend the long-term behavioral effects observed. Rats were injected either once or twice with chlorpyrifos (CPF) and then tested months after in a two-trial repeated acquisition task in a water maze. Our results confirm and extend the long-term behavioral effects of subcutaneous administration of CPF. The two treatments used produced performance decrements that suggest functional central nervous system alterations.     

MR imaging of the labral-capsular complex: normal variations

Understanding the normal anatomy of the shoulder and its variations is important for the proper interpretation of MR images. This study was performed to describe variations in the normal labral-capsular complex as seen on MR images. MR images of 52 shoulders in 30 asymptomatic volunteers and 27 shoulders of symptomatic patients who had subsequent arthroscopy and/or reconstructive surgery were obtained with 1.5-T MR. The 52 scans of the asymptomatic group were reviewed by three radiologists in conference, and the assessment of labral shapes and capsular insertions was done by consensus. The 27 MR scans of the shoulders in the symptomatic group were reviewed by one radiologist before and after the asymptomatic cases were interpreted. Differences in these two interpretations were shown on receiver-operating-characteristic curves by using the results of subsequent arthroscopy and surgery as the gold standard. The anterior and posterior parts of the labra, respectively, varied in shape but showed several dominant features: triangular (45%, 73%), round (19%, 12%), cleaved (15%, 0%), notched (8%, 0%), flat (7%, 6%), and absent (6%, 8%). Most capsules inserted anteriorly on the labrum (47%) or glenoid rim (49%). All posterior insertions were on the labrum (100%). Intrinsic labral signal was noted on proton density-weighted images, but never on T2-weighted images. Receiver-operating-characteristic curves from interpretations of the symptomatic and clinical cases made before and after evaluation of the asymptomatic shoulders showed the interpretations improved considerably after scans of asymptomatic volunteers were studied. Our study reveals a wide variability in the MR appearance of the labral-capsular complex in asymptomatic shoulders.     

Sphincter injury after anal dilatation demonstrated by anal endosonography.

Anal dilatation is still used in the treatment of anal fissure and haemorrhoids. Using anorectal physiology and anal endosonography we have studied 12 men presenting with faecal incontinence following anal dilatation. Resting anal pressures were low, pudendal nerve latencies were normal; 11 men had a disrupted internal anal sphincter and in ten this was extensively fragmented. Three also had defects of the external anal sphincter. These findings demonstrate for the first time the nature of the structural injury which may be caused by anal dilatation.     

Extracellular characteristics of putative cholinergic neurons in the rat laterodorsal tegmental nucleus

The extracellular electrophysiological properties of neurons in the laterodorsal tegmental nucleus (LDT), a major source of cholinergic afferents to the thalamus, were studied in chloral hydrate-anesthetized rats. A combination of antidromic activation from the thalamus and histological verification of recording sites was used to correlate the identity of extracellular recordings in the rat LDT with cholinergic neurons in that region. All neurons antidromically activated by stimulation of the anteroventral thalamus were histologically verified to be within clusters of cholinergic (NADPH-d-positive) cells in the LDT or in the adjacent nucleus locus coeruleus (LC). The thalamically projecting LDT neurons had a homogeneous neurophysiological profile consisting of long duration action potentials (mean = 2.5 ms), slow conduction velocities (mean = 0.78 m/s), and lengthy chronaxie values (mean = 0.725 ms). The appearance and axonal characteristics of these neurons resembled those of noradrenergic LC neurons, but the two populations exhibited substantially different spontaneous activity patterns and sensory responsiveness. These characteristics may be useful in the preliminary identification of putative cholinergic neurons in vivo, and thereby provide a foundation for exploring the neuropharmacology, afferent modulation, sensory responsiveness and behavioral correlates of the brainstem cholinergic system.     

The precision of positron emission tomography: theory and measurement

The limits of quantitation with positron emission tomography (PET) are examined with respect to the noise propagation resulting from radioactive decay and other sources of random error. Theoretical methods for evaluating the statistical error have been devised but seldom applied to experimental data obtained on human subjects. This paper extends the analysis in several ways: (1) A Monte Carlo method is described for tracking the propagation of statistical error through the analysis of in vivo measurements; (2) Experimental data, obtained in phantoms, validating the Monte Carlo method and other methods are presented; (3) A difference in activation paradigm, performed on regional CBF (rCBF) data from five human subjects, was analyzed on 1.6-cm diameter regions of interest to determine the mean fractional statistical error in PET tissue concentration and in rCBF before and after stereotactic transformation; and (4) A linear statistical model and calculations of the various statistical errors were used to estimate the magnitude of the subject-specific fluctuations under various conditions. In this specific example, the root mean squared (RMS) noise in flow measurements was about three times higher than the RMS noise in the concentration measurements. In addition, the total random error was almost equally partitioned between statistical error and random fluctuations due to all other sources.