Initiation of humoral and cellular immune responses in patients with refractory Hodgkin's disease by treatment with an anti-CD16/CD30 bispecific antibody

Fifteen patients with refractory Hodgkin's disease were treated in a dose-escalation trial with the bispecific monoclonal antibody (bi-mAb) HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkin's-associated CD30 antigen. Treatment consisted of four cycles of four bi-mAb infusions given over 1 h every 3–4 days at different dose levels ranging from 1 mg/m² to 64 mg/m². Measurable serum levels (above 0.1 μg/ml) of circulating bi-mAb could be detected in patients treated with doses above 4 mg/m², reaching peak levels of 9.5 μg/ml immediately after the end of antibody infusion on the highest dose level. Bi-mAb elimination corresponded to second-order kinetics with a terminal half-life time (t _1/2,β) of 28–32 h. Bi-mAb treatment induced the occurrence of human anti-(mouse Ig) antibodies (HAMA) in 6 out of 13 patients initially testing negative. All 6 patients not only developed anti-isotypic anti-(mouse Ig) but also anti-idiotypic and anti-anti-idiotypic antibodies. While no consistent changes of peripheral blood cell counts, or of any lymphocyte subpopulation including natural killer (NK) cells, has been observed, 4 out of 6 evaluable patients treated with doses of at least 4 mg/m² showed an increase of NK cell activity within 2 weeks after treatment, which lasted for a maximum of 12 weeks. Circulating amounts of soluble CD30 antigen could be detected in the serum of 6 patients. However, like the results and time courses of all the other immunological parameters evaluated, this was not predictive for treatment outcome. Received: 16 September 1999 / Accepted: 6 January 2000     

Analyzing Chromatin Structure and Transcription Factor Binding in Yeast

The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatinin vivo.The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cellsin vivoto determine the binding of factors tociselements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeastPHO8gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of atransactivator with an altered or perturbed chromatin organization at a specific locus.     

Role of PAR2 in murine pulmonary pseudomonal infection

Proteinases can influence lung inflammation by various mechanisms, including via cleavage and activation of protease-activated receptors (PAR) such as PAR2. In addition, proteinases such as neutrophil and/or Pseudomonas-derived elastase can disarm PAR2 resulting in loss of PAR2 signaling. Currently, the role of PAR2 in host defense against bacterial infection is not known. Using a murine model of acute Pseudomonas aeruginosa pneumonia, we examined differences in the pulmonary inflammatory response between wild-type and PAR2(-/-) mice. Compared with wild-type mice, PAR2(-/-) mice displayed more severe lung inflammation and injury in response to P. aeruginosa infection as indicated by higher bronchoalveolar lavage fluid neutrophil numbers, protein concentration, and TNF-alpha levels. By contrast, IFN-gamma levels were markedly reduced in PAR2(-/-) compared with wild-type mice. Importantly, clearance of P. aeruginosa was diminished in PAR2(-/-) mice. In vitro testing revealed that PAR2(-/-) neutrophils killed significantly less bacteria than wild-type murine neutrophils. Further, both neutrophils and macrophages from PAR2(-/-) mice displayed significantly reduced phagocytic efficiency compared with wild-type phagocytes. Stimulation of PAR2 on macrophages using a PAR2-activating peptide resulted in enhanced phagocytosis directly implicating PAR2 signaling in the phagocytic process. We conclude that genetic deletion of PAR2 is associated with decreased clearance of P. aeruginosa. Our data suggest that a deficiency in IFN-gamma production and impaired bacterial phagocytosis are two potential mechanisms responsible for this defect.     

Protein Disulfide Isomerase 2 of Chlamydomonas reinhardtii Is Involved in Circadian Rhythm Regulation

Protein disulfide isomerases （PDIs） are known to play important roles in the folding of nascent proteins and in the formation of disulfide bonds. Recently, we identified a PDI from Chlamydomonas reinhardtii （CrPDI2） by a mass spectrometry approach that is specifically enriched by heparin affinity chromatography in samples taken during the night phase. Here, we show that the recombinant CrPDI2 is a redox-active protein. It is reduced by thioredoxin reductase and catalyzes itself the reduction of insulin chains and the oxidative refolding of scrambled RNase A. By immunoblots, we confirm a high-amplitude change in abundance of the heparin-bound CrPDI2 during subjective night. Interestingly, we find that CrPDI2 is present in protein complexes of different sizes at both day and night. Among three identified interac- tion partners, one （a 2-cys peroxiredoxin） is present only during the night phase. To study a potential function of CrPDI2 within the circadian system, we have overexpressed its gene. Two transgenic lines were used to measure the rhythm of phototaxis~ In the transgenic strains, a change in the acrophase was observed. This indicates that CrPDI2 is involved in the circadian signaling pathway and, together with the night phase-specific interaction of CrPDI2 and a peroxiredoxin, these findings suggest a close coupling of redox processes and the circadian clock in C. reinhardtii.     

Left Ventricular Systolic Dysfunction in Asymptomatic Marfan Syndrome Patients Is Related to the Severity of Gene Mutation: Insights from the Novel Three Dimensional Speckle Tracking Echocardiography

Background

In asymptomatic Marfan syndrome (MFS) patients we evaluated the relationship between the types of fibrillin-1 (FBN1) gene mutation and possible altered left ventricular (LV) function as assessed by three-dimensional speckle tracking echocardiography (3D-STE).

Methods and Results

Forty-five MFS patients (mean age 24±15 years) and 40 age-matched healthy controls were studied. Genetic evaluation for the FBN1 gene was carried on 32 MFS patients. Gene mutation (n = 15, 47%) was classified as mild when the mutation resulted in nearly normally functioning protein, while mutations resulting in abnormally function protein were considered to be severe (n = 17, 53%). All patients and controls underwent 3D-STE for evaluation of LV function by an echocardiographer blinded to the results of the genetic testing. Compared to controls, MFS patients had significantly lower 3D-STE derived LV ejection fraction (EF, 57.43±7.51 vs. 62.69±4.76%, p = 0.0001), global LV longitudinal strain (LS, 14.85±2.89 vs. 17.90±2.01%, p = 0.0001), global LV circumferential strain (CS, 13.93±2.81 vs. 16.82±2.17%, p = 0.0001) and global LV area strain (AS, 25.76±4.43 vs. 30.51±2.61%, p = 0.0001). Apart from the global LV LS all these parameters were significantly lower in patients with severe gene mutation than in those with mild mutation (p<0.05). In the multivariate linear regression analysis only the type of mutation had a significant influence on the 3D-STE derived LVEF (p = 0.017), global CS (p = 0.005) and global AS (p = 0.03).

Conclusions

In asymptomatic MFS patients latent LV dysfunction can be detected using 3D STE. The LV dysfunction is mainly related to the severity of gene mutation, suggesting possible primary cardiomyopathy in MFS patients.     

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.     

The route of passive chloride movement across amphibian skin: localization and regulatory mechanisms

Transepithelial Cl(-) conductance (G(Cl)) in amphibian skin can be activated in several species by serosa positive potentials. Mitochondria-rich cells (MRC) or tight junctions (TJ) between the epithelial cells are possible sites for this pathway. The properties and the techniques used to investigate this pathway are reviewed in the present paper. In situ techniques are preferable, since specific properties of the MRC are apparently not maintained in isolated cells. Volume measurements and electronprobe microanalysis of intracellular ions suggest the localization of voltage-activated G(Cl) to MRC. G(Cl) correlates poorly with the density of MRC. The vibrating voltage probe allows quantitative correlation of the local Cl(-) current through morphologically identified structures and the transepithelial Cl(-) current. Our analysis shows that 80% of the voltage-activated Cl(-) current is accounted for by current through MRC or their immediate vicinity. The activation patterns of this current and the inhibition by the alpha(1)-adrenergic agonist, epinephrine, conform to those of the transepithelial current. However, less than 20% of the MRC are active at a certain moment and the activity is spontaneously variable with time. The molecular nature of this pathway, physiological control mechanisms and their relation to the temporal activity of MRC remain to be studied.     

Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

We describe here a molecular genetic approach for imaging synaptic inhibition. The thy-1 promoter was used to express high levels of Clomeleon, a ratiometric fluorescent indicator for chloride ions, in discrete populations of neurons in the brains of transgenic mice. Clomeleon was functional after chronic expression and provided non-invasive readouts of intracellular chloride concentration ([Cl(-)](i)) in brain slices, allowing us to quantify age-dependent declines in resting [Cl(-)](i) during neuronal development. Activation of hippocampal interneurons caused [Cl(-)](i) to rise transiently in individual postsynaptic pyramidal neurons. [Cl(-)](i) increased in direct proportion to the amount of inhibitory transmission, with peak changes as large as 4 mM. Integrating responses over populations of pyramidal neurons allowed sensitive detection of synaptic inhibition. Thus, Clomeleon imaging permits non-invasive, spatiotemporally resolved recordings of [Cl(-)](i) in a large variety of neurons, opening up new opportunities for imaging synaptic inhibition and other forms of chloride signaling.