Suppression of in vitro IgM rheumatoid factor production by diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) in patients with refractory rheumatoid arthritis

OBJECTIVE: To investigate the effect of diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) on in vitro synthesis of immunoglobulin and rheumatoid factor (RF) in patients with severe refractory rheumatoid arthritis (RA) enrolled in a phase II, double blind, placebo controlled study. METHODS: Anticoagulated venous blood samples were obtained before (Day 1) and after (Day 28) intravenous infusion of either DAB 486IL-2 at 0.075 mg/kg/day (12 patients) or saline placebo (10 patients) on Days 1-5. Peripheral blood leukocytes (PBL) were prepared by density gradient centrifugation, cultured in the presence and absence of pokeweed mitogen (PWM) for one week, and culture supernatants assayed for immunoglobulins and IgM RF by ELISA. RESULTS: Compared to placebo treated patients, PWM induced IgM RF synthesis by PBL decreased after treatment with DAB 486IL-2 (p = 0.043). However, there was no apparent correlation with clinical improvement. PWM induced IgM, IgA, and IgG synthesis also tended to decrease, although the changes did not attain statistical significance. In contrast, PWM induced IgM RF, IgM, IgA, and IgG synthesis by PBL from patients treated with placebo tended to increase during the observation period. Spontaneous immunoglobulin and IgM RF production by PBL from either the DAB 486IL-2 or placebo patients remained stable. CONCLUSION: These observations raise the possibility that DAB 486IL-2 may diminish B cell function either directly or indirectly through effects on T cell function, but the change may not correspond to clinical response.     

Gas-Liquid chromatography of fatty derivatives. I. Separation of homologous series ofa-olefins,n-hydrocarbons,n-nitriles,and n-alcohols

Summary Gas-liquid chromatography has been shown to be applicable to the analysis of several fatty derivatives. The difficulties inherent in conventional separation schemes such as liquid-liquid chromatography, fractional distillation, and crystallization can be overcome through the use of gas-liquid chromatography. It becomes the preferred procedure for several reasons: a) fractions are more nearly pure, b) analysis time is a fraction of the time required for conventional separations, c) operational procedures are simple and highly reproducible, d) sample size requirements are low, e) the cost per unit analysis is low. These factors suggest that gas-liquid chromatography is highly useful as an analytical tool in fat research. In many cases the determination of chainlength distribution has already become a routine function. There remain areas in which gas-liquid chromatography has not been completely successful. Until very recently the separation of fatty methyl esters of similar chain length and varying degrees of unsaturation has been time-consuming and tedious. Several new partitioning agents hold out great promise in the separation of the isomeric C₁₈ fatty acids. More selective partitioning agents and refined techniques undoubtedly will equally improve the resolution of unsaturated alcohols and other unsaturated fatty derivatives. Presented at annual meeting, American Oil Chemists' Society, Memphis, Tenn., April 21–23, 1958. Technical Paper No. 160, Archer-Daniels-Midland Company.     

Subjects presented at ASHP Midyear Clinical Meetings, 1967-90

The subject matter and trends of presentations made at ASHP Midyear Clinical Meetings (MCMs) were studied. A computerized database of information from MCM and Annual Meeting (AM) program and abstract books was created. The data were analyzed to determine the distribution of MCM presentations by subject and by author for the period 1967 to 1990, determine if there were differences in subjects covered between MCMs and AMs, explore the proposition that there has been duplication of material in MCM presentations, and evaluate the frequency with which MCM presentations have been published in ASHP journals. The total number of presentations made at MCMs from 1967 through 1990 was 8180, while the total for the AMs was 1547 for the two periods (1962-71 and 1985-90) studied. The most common keywords in titles were "pharmacy," "drug," "patient," "hospital," and "service." All International Pharmaceutical Abstracts subject categories and two other categories were represented; presentations in the institutional pharmacy practice category were the most frequent, while pharmacognosy-related presentations were least frequent. The overwhelming number of authors made only one presentation and were listed as the first author. The subjects of presentations were similar between AMs and MCMs. A tendency toward duplication of material was found. Of the 8180 MCM presentations, at least 1005 were published in an ASHP journal. Between 1967 and 1990, presentations at MCMs covered a wide range of subjects but were sometimes duplicative or not on the cutting edge.     

Cytomechanics of axonal development

Mechanical tension is a robust regulator of axonal development of cultured neurons. We review work from our laboratory, using calibrated glass needles to measure or apply tension to chick sensory neurons, chick forebrain neurons, and rat PC12 cells. We survey direct evidence for two different regimes of tension effects on neurons, a fluid-like growth regime, and a nongrowth, elastic regime. Above a minimum tension threshold, we observe growth effects of tension regulating four phases of axonal development: 1. Initiation of process outgrowth from the cell body; 2. Growth cone-mediated elongation of the axon; 3. Elongation of the axon after synaptogenesis, which normally accommodates the skeletal growth of vertebrates; and 4. Axonal elimination by retraction. Significantly, the quantitative relationship between the force and the growth response is surprisingly similar to the simple relationship characteristic of Newtonian fluid mechanical elements: elongation rate is directly proportional to tension (above the threshold), and this robust linear relationship extends from physiological growth rates to far-above-physiological rates. Thus, tension apparently integrates the complex biochemistry of axonal elongation, including cytoskeletal and membrane dynamics, to produce a simple "force input/growth output" relationship. In addition to this fluid-like growth response, peripheral neurons show elastic behaviors at low tensions (below the threshold tension for growth), as do most cell types. Thus, neurites could exert small static forces without diminution for long periods. In addition, axons of peripheral neurons can actively generate modest tensions, presumably similar to muscle contraction, at tensions near zero. The elastic and force-generating capability of neural axons has recently been proposed to play a major role in the morphogenesis of the brain.     

Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.     

Clinicopathologic analysis of microscopically invasive breast carcinoma

Breast biopsy or mastectomy cases having diagnoses of carcinoma in situ with "microinvasion," "minimal invasion," "focal invasion," or "suggestive of invasion" were reviewed and all histologically identified foci of invasive disease from each case were measured using an ocular micrometer. Cases in which any single focus of invasion was greater than 5 mm or the added size of separate invasive foci exceeded 10 mm were excluded, resulting in a study group of 75 patients. Invasive neoplasm was present in the initial biopsy in 69 of 75 cases (92%); however, residual invasive neoplasm was found in the subsequent lumpectomy/mastectomy from 14 of these (20%). In 59% of cases, two or more histologically separate foci of invasion were identified. Invasive foci consisted of isolated cells or cell clusters, each less than 1 mm (microfocal invasion), in 33% of cases. In 12 cases, the sum of individual invasive foci was 5 to 10 mm. Axillary lymph nodes (LN) from 5 of 69 patients (7%) contained metastatic carcinoma (four cases, one LN positive; one case, two LN positive). The cumulative sizes of all invasive foci in the LN-positive group were microfocal invasion (one case), 0.6 mm (one case), 1.1 mm, 2.5 mm, and 5.8 mm. The difference in frequency of axillary node metastasis between tumors with microfocal and measurable invasion (4.3% v 8.6%) was not statistically significant. Follow-up data were available on 55 cases (mean interval, 66.1 months). One (node-negative) patient had duct carcinoma in situ recurrence in the same breast 4 years after initial treatment. Another (with unknown node status) developed an axillary lymph node metastasis 13 months after initial treatment (96% disease-free survival). We conclude that microscopic stromal invasion in breast carcinoma, at least in the setting of significant in situ component, is often initiated from multiple foci. Patients with microscopically invasive breast carcinoma have a small but significant risk of axillary metastases, although a highly favorable survival.     

Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed alpha/beta protein, Escherichia coli thioredoxin

Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins. The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (Fl) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state. Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degreesC) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.     

Pressure dependence of superconductivity in Ba4C60

In Ba_xC₆₀ superconductivity (sc) at T_c=(6.8±0.2) K is associated with the x=4 phase. Here we present pressure studies of T_c of a Ba_xC₆₀ sample (dT_c/dp= –1.9 K/GPa) which exhibits a large volume fraction of the x=4 phase. In the alkali-fullerides K₆₀C₆₀ and Rb₃C₆₀ T_c is governed by the electronic density of states N(E_F) (T_c exp{–1/N(E_F)V}). The dependence of N(E_F) on volume can be approximated by N(E_F)=N₀ d _eff ³, where d_eff is the surface to surface distance of neighboured C₆₀-molecules. This relationship is corroborated by (T_C,d)-data of various compounds of the form A_3–xB_xC₆₀ (A: K, Rb; B: K, Rb, Cs; x2). Strong deviation from this behavior is found for the alkaline-fulleride superconductor Ba₄C₆₀. This deviation is attributed to the hybridization of alkaline-metal s-, d-states and C₆₀ -states.     

The gradual threshold model of ambivalence: relating the positive and negative bases of attitudes to subjective ambivalence

Eighty subjects viewed and visually imagined upright or rotated alphanumeric characters and later judged whether test characters were previously seen or imagined (reality monitoring). Identification and test characters were presented verbally or visually. When characters were identified and tested verbally, source confusions (misjudging a seen character as "imagined" and vice-versa) were infrequent and were comparable for rotated and upright characters. When characters were identified and tested visually, source confusions were more frequent and were influenced by character rotation. Memories for imagined characters were especially susceptible to source confusion. Also source confusions for seen characters increased when characters were rotated. These results are consistent with the proposal that increasing sensory similarity between perceived and imagined items increases source confusion and that perceived rotation generates cognitive operations similar to those generated when the subject imagines a character rotated.     

Fast transmission CT for determining attenuation maps using a collimated line source, rotatable air-copper-lead attenuators and fan-beam collimation

We describe a technique using a line source and a rotatable air-copper-lead assembly to acquire gamma transmission computed tomographic (TCT) data for determining attenuation maps to compensate SPECT emission scans. The technique minimizes problems associated with discriminating 99mTc transmission and 201Tl emission photons and requires only a modest increase in total study time. A 99mTc line source and a stacked foil ("multislat") collimator are placed near the focal line of a fan-beam collimator (114 cm focal length) mounted on one detector of a triple-camera SPECT system. We acquired TCT data of plastic rod and anthropomorphic thorax phantoms to investigate the capability of the line source and rotatable air-copper-lead attenuators to determine attenuation maps. The data were acquired with and without 5.4 MBq (145 microCi) of 201Tl placed in the myocardial chamber of the thorax phantom. Phantoms also were scanned using a curved transmission slab source mounted to a parallel-hole collimator. Fan-beam TCT images have improved resolution compared with parallel-beam TCT images. Two patient scans also were performed to evaluate the clinical usefulness of fan-beam TCT. The rotatable air-copper-lead attenuator method eliminates contamination of emission data by transmission photons and reduces spill-over of emission data into the transmission energy window for some cases. Results show the feasibility of using fast, sequential or interlaced transmission scans of a line source within a rotatable air-copper-lead attenuator assembly to obtain accurate attenuation maps for SPECT attenuation compensation.