Multiple-antigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens.

Antibody phage display is a powerful tool for the generation of monoclonal antibodies against virtually any given antigen. Chickens are phylogenetically more distant from humans compared to other laboratory animals, such as mice and rats. Therefore, the use of chickens is especially beneficial when generating recombinant antibodies against human autoantigens, which are often highly conserved among mammals. Another advantage of using chickens in antibody phage display is that the preparation of single chain variable fragment (scFv) antibody libraries is faster and easier compared to preparing such libraries from other species, as only two primer sets are needed for amplification of the chicken variable heavy chain (V(H)) and variable light chain (V(L)) genes. In the present study we explored the possibility to immunize chickens with antigen cocktails for the generation of recombinant antibody fragments directed to a range of human autoantigens. Two pairs of chickens were immunized with two cocktails of seven recombinant autoantigenic proteins, libraries were prepared and panned on the individual proteins. The polyclonal chicken sera reacted strongly with most of the antigens used for immunization. By creating and screening single-chain variable fragment antibody phage display libraries, recombinant monoclonal antibody fragments were isolated successfully against the autoantigens annexin XI, centromere protein B, heat shock protein B3, DNA topoisomerase I, histidyl tRNA synthetase, Ro52, Ro60, Rpp30 and U1A. In conclusion, the immunization of only four chickens with two distinct pools of a total of 14 autoantigenic proteins allowed the isolation of scFvs against nine of these antigens.

Apoptotic modifications affect the autoreactivity of the U1 snRNP autoantigen.

A hallmark of systemic autoimmune diseases is the presence of high titers of serum autoantibodies targeting a diversity of autoantigens. Most components of the U1 snRNP complex are autoantigenic in systemic lupus erythematosus (SLE) and SLE overlap syndrome, which is also called mixed connective tissue disease (MCTD). It is hypothesized that posttranslational modifications, in particular cell death-associated modifications, play an important role in breaking tolerance to self-antigens. Recently, it became clear that the U1 snRNP particle, more specifically its U1-70K protein component, displays a new epitope during apoptosis. This review intends to give an overview of the modifications that occur on the U1 snRNP autoantigens, especially those arising during cell death, to summarize recent data describing autoantibody reactivities with apoptosis-specific epitopes on the U1 snRNP complex, and to provide some insight into the mechanisms that might underlie the immune response to self-antigens.

Epidemiology of apparent life threatening events.

AIMS: To investigate the epidemiology and risk factors of apparent life threatening events (ALTE). METHODS: A prospective study enrolled all live-born infants in the Tyrol (1993-2001). Information on pregnancy, sociodemographic characteristics, child care practices, and infant's behaviour in the first four to six weeks of life was collected with a standardised questionnaire, and was available for 44,184 infants. ALTE was identified from hospital admission records. RESULTS: During the study period 164 ALTE cases were identified, corresponding to an incidence of 2.46/1000 live births. In 73 of these infants no cause for the event and no comorbidity could be found (idiopathic ALTE). On average ALTE manifested ten weeks earlier than SIDS. Of various SIDS risk factors in the survey area, the prone sleeping position, smoking during pregnancy, low gestational age, profuse night sweating, and family history of infant death showed a moderate relation to the risk of overall ALTE, but only smoking maintained significance in the multivariate risk model. None of these variables was associated with idiopathic ALTE. In contrast to SIDS the frequency of ALTE did not change during the study period. None of the ALTE infants experienced SIDS later in life. Behavioural abnormalities such as feeding difficulties, episodes of pallor, cyanotic episodes, and repeated apnoea episodes were strongly associated with an increased risk of overall and idiopathic ALTE. CONCLUSIONS: Although there are some similarities in the clinical presentation and epidemiology of SIDS and ALTE, differences clearly predominate. Accordingly, ALTE and SIDS should not be considered different manifestations of the same disease process.

Prepidil compared to Propess for cervical ripening.

OBJECTIVES: To compare the efficacy and safety of intracervical PGE(2) gel (Prepidil) with intravaginal PGE(2) insert (Propess) in cervical ripening and induction of labour. STUDY DESIGN: This was a retrospective analysis of patients with single pregnancies, intact membranes and a viable fetus who required cervical ripening. In the first half of 1998, all patients received Prepidil and in the first half of 1999 all patients received Propess. In both periods 50 consecutive files were retrieved and compared. RESULTS: The Prepidil and Propess group were similar in patient characteristics, indication for induction of labour and delivery outcome. Cervical ripening within 24 h was achieved in the Propess group in 80% as compared to 56% in the Prepidil group. The application-membrane rupture-interval was shorter in the Propess group compared to the Prepidil group (23.7 +/- 21.3 h versus 56.4 +/- 77.7 h, P = 0.009) as well as the application-delivery interval (29.8 +/- 22.0 h versus 62.0 +/- 78.8 h, P = 0.039). In the Propess group 62% delivered within 24 h compared to 28% in the Prepidil group. A smaller number of applications was observed in the Propess group (1.2 versus 1.8, P < 0.005). Both methods proved to be safe in cervical priming and no relevant differences in mode of delivery and outcome of labour were detected. None of the patients required an emergency caesarean section during the treatment with prostaglandines. CONCLUSIONS: Both prostaglandin E(2) agents are safe and effective in achieving cervical ripening; however the vaginal insert demonstrated a shorter application-delivery interval. The hospital stay was consequently shorter, contributing to cost effectiveness.

The flaginserter: a simple device for automatically marking events in video recordings.

Dynamic imaging of cellular responses often involves the use of standard video components. While these components (cameras, monitors, etc.) are relatively inexpensive and easily available, there is the difficulty of integrating them into experimental set-ups. Especially for video recordings of fast signals that occur in neurobiological preparations (e.g. synaptic responses), there is a critical need for accurate synchronisation of video images with external events, such as extracellular stimulation or intracellular depolarising voltage steps. We developed a circuit that allows such events within the video sequence to be marked by means of a white flag that is inserted into the video image. Depending on the mode of insertion, the time resolution is better than 17 ms or better than 65 micros (i.e. a single video field or video line, respectively). The circuit has been shown to work reliable in combination with video-rate confocal imaging and patch-clamp recordings.

Development of a scanning, solar-blind, water Raman lidar.

The need for an instrument capable of measuring water-vapor fluxes over mixed canopy and large areas has long been recognized. Such a device would greatly enhance the study of evapotranspiration processes and has great practical value for water management. To address this problem, a scanning water Raman lidar has been designed and constructed. Analytical methods have also been developed to take advantage of the type of information that this lidar can generate. The lidar is able to measure the absolute water content and calculate the evaporative flux quickly over relatively large areas. This capability provides new opportunities for the study of microscale atmospheric processes. The variogram data indicate that the spatial sampling size must be of the order of 10 m if fluxes and scalars are to be properly represented. Examples of data are presented.

The dual effect of internal tetramethylammonium ions on the open states of the sodium channel in the squid giant axon.

Voltage-clamp recordings of INa in squid axons dialysed with Cs or TMA, and bathed in low Na choline seawater, showed that, except close to threshold, the initial peak of fast-inactivating current was invariably decreased by TMA, whereas the non-inactivating current in the steady state was simultaneously increased. The results suggest that although TMA does not act directly on the movements of the voltage sensors that activate the sodium system, it blocks single-channel conductance in a voltage-dependent fashion in both the open states of the Na channel, while it has an entirely different type of action by increasing the probability of late openings in the steady state. Another difference between the two open states was that the sodium permeability coefficient had a Q10 of 1.8 in the initial open state, whereas in the steady state the effect of temperature was much smaller or even negative.

A pulse generating and data recording system based on the microcomputer PDP 11/23.

A widely applicable measuring system, especially suited for all types of 'voltage-clamp' (including 'patch-clamp') experiments, is presented. It is mainly based on commercially available hardware and software components. All interfacing with the analog world takes place on the front panel of the microcomputer box. Synchronization of pulse generation with data acquisition and other control actions take advantage of the four upper bits not used by the digital-to-analog converter during direct memory access transfer.

Successful nonsurgical treatment of tuberculous empyema in an irreducible pleural space.

A 78-year-old woman with empyema due to Mycobacterium tuberculosis in a chronic pleural space was successfully treated with a 24-month course of oral isoniazid, rifampin, ethambutol, and serial space-emptying thoracocenteses. Besides dramatic clinical improvement, follow-up pleural fluid analyses demonstrated gradual replacement of the empyema with a sterile pleural exudate, which has persisted 24 months after cessation of therapy. This case demonstrates a therapeutic program that was an effective alternative to decortication or thoracoplasty for tuberculous empyema in an irreducible pleural space.

Modification of ultraviolet radiation effects on the membrane of myelinated nerve fibers by sulfhydryl compounds.

The modification of the ultraviolet blocking of sodium channels and of the ultraviolet-induced potential shift of the gating parameters by means of the sulfhydryl compounds l-cysteine and 2-mercaptoethanol was investigated in the node of Ranvier under voltage-clamp conditions. The UV wavelength was 280 nm. The radiation-induced potential shift of the voltage-dependent gating parameters was prevented or even reversed by the action of the sulfhydryl compounds (internal application), while the blocking effect was not affected. It is concluded that the two radiation effects are caused by two separate photoreactions. Internally applied N-ethylmaleimide, binding specifically to protein-SH groups, exhibits an effect similar to the ultraviolet-induced potential shift, without affecting the maximum sodium permeability. Therefore, the ultraviolet-induced potential shift might be caused by a photocatalyzed oxidation of -SH groups of membrane proteins changing the surface charge density at the inner side of the nodal membrane.

Single Na channels in mouse neuroblastoma cell membrane. Indications for two open states.

Single sodium channel currents were studied in mouse neuroblastoma cells. Channel conductance (gamma) was 11.6 +/- 3.4 pS at 6-8 degrees C. Unitary current amplitudes and channel activity increased with increasing temperature. The Arrhenius plot of the conductance was linear between 5 and 35 degrees C with an activation enthalpy of 27.1 kJ/mole (Q10 = 1.28). Amplitude distributions were fitted by the sum of two Gaussian functions indicating the presence of two different single channel amplitudes: smaller i1 and larger i2. The relative probability of appearance of i1, which has a shorter mean open time, was higher during the early phase of depolarization (t less than 16 ms). The open time histograms, inactivating phase of macroscopic currents and delay time histograms were fitted by the sum of two exponentials. The distinct kinetic and steady-state parameters reflect two open states of sodium channels.

Changes of ultraviolet sensitivity of voltage-clamped sodium channels during their potential-induced conductance cycle.

The effects of ultraviolet (UV) radiation at various intermediate states of excitation of the frog nerve membrane were measured under voltage-clamp conditions. By synchronizing a UV flash source (flash duration: 10 microseconds) with the stimulating voltage-clamp pulses it was possible to irradiate the membrane at any state of excitation. The resulting UV sensitivities gamma Na and gamma h of gNa and h infinity (V = 0) were compared with those found at the end of a hyperpolarizing prepulse (m = 0, h = 1). The irradiation always induced a decrease of both parameters, gNa and h infinity (V = 0), and the more "h-gates" there were in the closed position at the moment of irradiation, the more pronounced were the effects. The normalized sensitivity of both parameters can be described fairly well by linear relations of the form: gamma x(m,h)/ gamma x(0, 1) = 1 + Px(1 - h) + Qxm2h (x = Na or h). The coefficients P and Q depend on wavelength. The results suggest a conformational change of the nodal membrane during excitation and an interdependence of m and h to some extent.

Phagocytosis by human alveolar macrophages and neutrophils: qualitative differences in the opsonic requirements for uptake of Staphylococcus aureus and Streptococcus pneumoniae in vitro.

The opsonic requirements for uptake of 3H-adenine-labeled Staphylococcus aureus (strains 502A, Wood 46, or 209) or Streptococcus pneumoniae (Type VII) by human alveolar macrophages (AM) or neutrophils (PMN) were evaluated and compared using a quantitative assay of phagocytosis. AM readily engulfed S. aureus organisms preopsonized with 1% albumin, serum, or serum that had been heated (56 degrees C for 30 min) to inactivate complement, whereas uptake of S. aureus by PMN occurred only after preopsonization with unheated, complement replete serum (2.5%). In contrast, uptake of S. pneumoniae organisms by AM or PMN did not occur with only albumin preopsonization, but required high concentrations (more than 40%) of unheated serum. The results indicate that there are qualitative and quantitative differences in the requirements for optimal ingestion of bacteria by human AM or PMN that relate not only to the type of organism but also to the type of phagocyte. Variations in opsonic requirements may be a key factor in the different cellular responses and clearances of inhaled S. aureus or S. pneumoniae organisms by the lungs.

Spontaneous regression of diffuse tracheobronchial amyloidosis.

Diffuse tracheobronchial amyloidosis which underwent an extensive spontaneous regression is described in a 39-year-old man. This regression was documented by serial roentgenograms of the chest, studies of pulmonary function, and bronchoscopic procedures with mucosal biopsies. The clinical implications of this spontaneous change are discussed.