Autologous lymphokine-activated killer cell therapy of Epstein-Barr virus-positive and -negative lymphoproliferative disorders arising in organ transplant recipients

Lymphoreticular malignancies, collectively called posttransplant lymphoproliferative disorders (PTLD), eventually develop in 2-5% of organ transplant recipients. They frequently undergo regression when immunosuppression is reduced or stopped. This feature has been associated with a previous or de novo Epstein-Barr virus (EBV) infection. We herein describe immunotherapy with autologous lymphokine-activated killer (LAK) cells in seven patients with PTLD (four EBV-positive patients and three EBV-negative patients). Autologous peripheral blood mononuclear cells were obtained by leukapheresis, depleted of monocytes, and cultured in the presence of interleukin 2 for 10 to 11 days. A single dose of 5.2 x 10(9) to 5.6 x 10(10) LAK cells was given intravenously. Systemic interleukin 2 was not administered. The four patients with EBV+ PTLD had complete tumor regression; two of them developed controllable rejection. Three patients are well 13-16 months after treatment; the fourth patient died of pneumonia 41 days after infusion. Three patients with EBV- lymphomas had no response despite prior evidence that their tumors also were subject to immune surveillance. Two of these three patients died after being given other treatment, and the third patient has persistent tumor. In conclusion, autologous LAK cell infusion was effective for treatment of four EBV+ organ transplant recipients. LAK cell efficacy for three patients with EBV- PTLD was not evaluable under the management circumstances in which this treatment was utilized.     

Double-blind randomized placebo-controlled study of homoeopathic prophylaxis of migraine

Homoeopathic remedies for migraine are widely available over the counter, statutorily offered by the national health service in the UK, and apparently popular with patients. Do they work? Sixty-three outpatients with migraine with or without aura by IHS criteria entered a 4-month randomized placebo-controlled, double-blind parallel-groups trial of individualized homoeopathic prophylaxis, the first month being baseline with all patients on placebo. Three patients (4.8%) dropped out, leaving 30 in each treatment group. There were chance differences in attack frequency and severity between the groups at baseline (attacks were more frequent but less severe in the placebo group). Both groups improved on therapy, but neither to a great extent on the primary outcome measure of attack frequency (verum: -19%; placebo: -16%). Reduction was mostly in mild attacks on placebo, more in moderate and severe attacks on homoeopathy. Few adverse events were reported. Overall, there was no significant benefit over placebo of homoeopathic treatment. The course of change differed between groups, and suggested that improvement reversed in the last month of treatment on placebo. On this evidence we cannot recommend homoeopathy for migraine prophylaxis, but cannot conclude that it is without effect.     

Outcome of blunt thoracic aortic injury in a level I trauma center: an 8-year review

BACKGROUND: The purpose of this study was to evaluate our experience with blunt thoracic aortic injury and identify factors predictive of outcome. METHODS: Hospital charts, trauma registry data, and autopsies of 64 patients with blunt thoracic aortic injury from 1988 to 1995 were reviewed. RESULTS: Patients were identified and segregated based on admission physiology. Group 1 patients (n = 19) arrived in arrest. Group 2 patients (n = 10) arrived in shock with systolic BP 90. Group 3 patients (n = 35) arrived with systolic BP>90. All patients in groups 1 and 2 expired. Injury Severity Scores for nonsurvivors in group 3 (n = 12) were significantly higher than survivors. There were no significant differences when comparing time of injury to repair or arrival between groups, or in mortality or paralysis comparing repair techniques or clamp/bypass times. Double lumen endotracheal tubes caused significant operative delays compared to single lumen tubes. CONCLUSIONS: Predictors of survivability were hemodynamic stability on arrival and lower Injury Severity Scores. In thoracic aortic injury patients arriving hemodynamically stable, Injury Severity Score correlated with mortality but not paralysis.     

Dopamine D3 receptor mutant mice exhibit increased behavioral sensitivity to concurrent stimulation of D1 and D2 receptors

The dopamine D3 receptor is expressed primarily in regions of the brain that are thought to influence motivation and motor functions. To specify in vivo D3 receptor function, we generated mutant mice lacking this receptor. Our analysis indicates that in a novel environment, D3 mutant mice are transiently more active than wild-type mice, an effect not associated with anxiety state. Moreover, D3 mutant mice exhibit enhanced behavioral sensitivity to combined injections of D1 and D2 class receptor agonists, cocaine and amphetamine. However, the combined electrophysiological effects of the same D1 and D2 agonists on single neurons within the nucleus accumbens were not altered by the D3 receptor mutation. We conclude that one function of the D3 receptor is to modulate behaviors by inhibiting the cooperative effects of postsynaptic D1 and other D2 class receptors at systems level.     

Dissecting the order of bacteriophage T4 DNA polymerase holoenzyme assembly

Most biological organisms rely upon a DNA polymerase holoenzyme for processive DNA replication. The bacteriophage T4 DNA polymerase holoenzyme is composed of the polymerase enzyme and a clamp protein (the 45 protein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme. The 45 protein must be loaded onto DNA by a clamp loader ATPase complex (the 44/62 complex). In this paper, the order of events leading to holoenzyme formation is investigated using a combination of rapid-quench and stopped-flow fluorescence spectroscopy kinetic methods. A rapid-quench strand displacement assay in which the order of holoenzyme component addition is varied provided data indicating that the rate-limiting step in holoenzyme assembly is associated with the clamp loading process. Pre-steady-state analysis of the clamp loader ATPase activity demonstrated that the four bound ATP molecules are hydrolyzed stepwise during the clamp loading process in groups of two. Clamp loading was examined with stopped-flow fluorescence spectroscopy from the perspective of the clamp itself, using a site-specific, fluorescently labeled 45 protein. A mechanism for T4 DNA polymerase holoenzyme assembly is proposed in which the 45 protein interacts with the 44/62 complex leading to the hydrolysis of 2 equiv of ATP, and upon contacting DNA, the remaining two ATP molecules bound to the 44/62 complex are hydrolyzed. Once all four ATP molecules are hydrolyzed, the 45 protein is poised on DNA for association with the polymerase to form the holoenzyme.     

Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.     

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint

OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.     

Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter

Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.