Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3

The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., W?lle, J., Plückthun, A., and Virnek?s, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.     

Role of depolarization and calcium in contractions of canine trachealis from endogenous or exogenous acetylcholine

The relationships of the electrical to the mechanical responses of the canine trachealis muscle during stimulation of its cholinergic nerves or exposure to exogenous acetylcholine were recorded in the single or the double sucrose gap. At 27 degrees C, the responses to a train of stimuli consisted of a transient depolarization excitatory junction potential of 10-30 mV followed by fading oscillations and contractions. When stimulus parameters were varied in the single sucrose gap, contractions were more closely associated with the occurrence of and varied in duration with the oscillations rather than with the amplitude of the EJP. Acetylcholine superfused at a concentration of 10(-6) M for 30 s caused a prolonged depolarization of 10-20 mV, but a much larger contraction than could be elicited by nerve stimulation. None of the responses to acetylcholine was significantly affected by the Ca channel antagonists, nifedipine, nitrendipine, or verapamil in Ca channel blocking concentrations. When tissues were exposed to a Ca-free medium, the excitatory junction potentials and oscillations rapidly disappeared, but the electrical and mechanical responses to acetylcholine persisted and only gradually disappeared with repetitive exposures. Furthermore, in a medium with normal Ca2+ in the double sucrose gap, depolarization by 10-15 mV with an applied current caused no contraction, and repolarization to the normal membrane potential during acetylcholine-induced contraction caused no relaxation. Tetraethylammonium ion (20 mM) depolarized the membrane, increased membrane resistance, and enhanced the secondary oscillations and contractions after field stimulation. No other K(+)-channel blocker tested (Ba2+, apamin, 4-aminopyridine, glibenclamide, charybdotoxin) had the effect of prolonging secondary oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)     

New role for Agrin in T cells and its potential importance in immune system regulation

Agrin plays a crucial role in the maintenance of the neuromuscular junction. However, it is expressed in other tissues as well, including T lymphocytes, where cell activation induces its expression. Agrin from activated T cells has the capacity to induce aggregation of key receptors and to regulate signalling. Interestingly, T cells isolated from patients with systemic lupus erythematosus over-express Agrin and its co-stimulation with the T cell receptor enhances production of pathogenic cytokines. These early studies point to an important function for Agrin in T cell biology and make the case for a more thorough and systematic investigation into its role in the immune system.     

Molecular relationships in Genista L. Sect. Spartocarpus Spach (Fabaceae)

 A molecular investigation on the species of Genista belonging to sect. Spartocarpus was undertaken with the aim of inferring phylogenetic relationships within the section. The combined cladistic analysis of ITS 1 and 2 and of the chloroplast DNA trnL^(UAA) intron yielded a single most parsimonious cladogram with G. aetnensis as sister group to the rest of the section, which is in turn divided into two major clades, one of which includes G. nissana, G. aucheri, G. sessilifolia, G. spartioides and G. haenseleri, and the other the remaining species of the section. This latter group has G. holopetala and G. radiata forming a clade, which is a sister group to a not entirely resolved monophylum including the G. ephedroides species complex. The latter shows a basal collapse of G. cilentina, G. dorycnifolia and G. numidica, and then a clade including G. ephedroides and G. valsecchiae, as well as a collapsed monophylum with G. demarcoi, G. gasparrini and G. thyrrena. The clade including the G. ephedroides species group has the lowest within-group molecular variation and appears to be recent in origin, as compared to the rest of the section. The pattern of cladogeny in the section seems to be related to geographical isolation, and is broadly congruent with palynological and biogeographical evidence. Received March 3, 2001 Accepted October 8, 2001     

Primary human CD4+ T cells have diverse levels of membrane lipid order that correlate with their function

Membrane lipid microdomains (lipid rafts) play an important role in T cell function by forming areas of high lipid order that facilitate activation. However, their role in regulating T cell differentiation and function remains controversial. In this study, by applying a new approach involving microscopy and flow cytometry, we characterize membrane lipid order in ex vivo primary human CD4(+) T cells. We reveal that differential membrane lipid order dictates the response to TCR stimulation. T cells with high membrane order formed stable immune synapses and proliferated robustly, intermediate order cells had reduced proliferative ability accompanied by unstable immune synapse formation, whereas low order T cells were profoundly unresponsive to TCR activation. We also observed that T cells from patients with autoimmune rheumatic disease had expanded intermediate order populations compared with healthy volunteers. This may be important in dictating the nature of the immune response since most IFN-γ(+)CD4(+) T cells were confined within intermediate membrane order populations, whereas IL-4(+)CD4(+) T cells were contained within the high order populations. Importantly, we were able to alter T cell function by pharmacologically manipulating membrane order. Thus, the results presented from this study identify that ex vivo CD4(+) T cells sustain a gradient of plasma membrane lipid order that influences their function in terms of proliferation and cytokine production. This could represent a new mechanism to control T cell functional plasticity, raising the possibility that therapeutic targeting of membrane lipid order could direct altered immune cell activation in pathology.     

Novel Strategies for Enhanced Removal of Persistent Bacillus anthracis Surrogates and Clostridium difficile Spores from Skin

Background

Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe^®) would reduce the burden of spores on skin.

Methods

Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths.

Results

Spore removal from hands was enhanced with Vashe soak (>2.5 log₁₀ reduction) versus soap and water wash or soak (~2.0 log₁₀ reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log₁₀ spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin.

Conclusions

Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin.     

Detection of salinity by the lobster, Homarus americanus.

Changes in the heart rates of lobsters (Homarus americanus) were used as an indicator that the animals were capable of sensing a reduction in the salinity of the ambient seawater. The typical response to a gradual (1 to 2 ppt/min) reduction in salinity consisted of a rapid increase in heart rate at a mean threshold of 26.6 +/- 0.7 ppt, followed by a reduction in heart rate when the salinity reached 22.1 +/- 0.5 ppt. Animals with lesioned cardioregulatory nerves did not exhibit a cardiac response to changes in salinity. A cardiac response was elicited from lobsters exposed to isotonic chloride-free salines but not to isotonic sodium-, magnesium- or calcium-free salines. There was little change in the blood osmolarity of lobsters when bradycardia occurred, suggesting that the receptors involved are external. Furthermore, lobsters without antennae, antennules, or legs showed typical cardiac responses to low salinity, indicating the receptors are not located in these areas. Lobsters exposed to reductions in the salinity of the ambient seawater while both branchial chambers were perfused with full-strength seawater did not display a cardiac response until the external salinity reached 21.6 +/- 1.8 ppt. In contrast, when their branchial chambers were exposed to reductions in salinity while the external salinity was maintained at normal levels, changes in heart rate were rapidly elicited in response to very small reductions in salinity (down to 29.5 +/- 0.9 ppt in the branchial chamber and 31.5 +/- 0.3 ppt externally). We conclude that the primary receptors responsible for detecting reductions in salinity in H. americanus are located within or near the branchial chambers and are primarily sensitive to chloride ions.     

G6PD Mediterranean accounts for the high prevalence of G6PD deficiency in Kurdish Jews

The Jews of Kurdistan are a small inbred population with a high incidence of -thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Recently, it was reported that the -thalassaemia in this population shows an unusual mutational diversity; 13 different mutations were identified, of which 4 had not previously been observed in any other population. In contrast, we now report that the G6PD deficiency, which has the highest known incidence in the world, and which affects about 70% of males, is almost entirely attributable to a single widespread mutation, G6PD Mediterranean.     

Lipid rafts and T-lymphocyte function: implications for autoimmunity

Experimental evidence indicates that the mammalian cell membrane is compartmentalized. A structural feature that supports membrane segmentation implicates assemblies of selected lipids broadly referred to as lipid rafts. In T-lymphocytes, lipid rafts are implicated in signalling from the T-cell antigen receptor (TCR) and in localization and function of proteins residing proximal to the receptor. This review summarizes the current literature that deals with lipid raft involvement in T-cell activation and places particular emphasis in recent studies investigating lipid rafts in autoimmunity. The potential of lipid rafts as targets for the development of a new class of immune-modulating compounds is discussed.     

Chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress

Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current (Isc), conductance (G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in+/+but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in+/+rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.