Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.

BACKGROUND: IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes. METHODS: IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm. RESULTS: BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap. CONCLUSION: Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition.

[Governmental batch sample testing of allergen products]. / Staatliche Chargenprüfung von Allergenpräparaten.

Allergen products for specific immunotherapy of type I allergies were first authorized for the German market in the 1970s. In addition to finished products manufactured in advance and in batches, so-called named patient products have recently been defined as Medicinal Products by the German Medicinal Products Act ("Arzneimittelgesetz", AMG 14th Revision 2005). Some allergen products previously marketed as named patient products are now required to obtain marketing authorization according to the German ordinance for therapy allergens. Products have to be batch released by the competent German Federal Agency, the Paul-Ehrlich-Institut (PEI). Samples of product batches are delivered to the PEI in order to perform experimental quality controls. With regard to named patient products, PEI tests batch samples of the bulk extract preparations used for manufacturing of the respective, named patient products. The institute releases approximately 2,800 allergen product batches annually.

Constructing an RNA world.

A popular theory of life's origins states that the first biocatalysts were not made of protein but were made of RNA or a very similar polymer. Experiments are beginning to confirm that the catalytic abilities of RNA are compatible with this 'RNA world' hypothesis. For example, RNA can synthesize short fragments of RNA in a template-directed fashion and promote formation of peptide, ester and glycosidic linkages. However, no known activity fully represents one presumed by the 'RNA world' theory, and reactions such as oxidation and reduction have yet to be demonstrated. Filling these gaps would place the hypothesis on much firmer ground and provide components for building minimal forms of RNA-based cellular life.

Isolation of new ribozymes from a large pool of random sequences [see comment].

An iterative in vitro selection procedure was used to isolate a new class of catalytic RNAs (ribozymes) from a large pool of random-sequence RNA molecules. These ribozymes ligate two RNA molecules that are aligned on a template by catalyzing the attack of a 3'-hydroxyl on an adjacent 5'-triphosphate--a reaction similar to that employed by the familiar protein enzymes that synthesize RNA. The corresponding uncatalyzed reaction also yields a 3',5'-phosphodiester bond. In vitro evolution of the population of new ribozymes led to improvement of the average ligation activity and the emergence of ribozymes with reaction rates 7 million times faster than the uncatalyzed reaction rate.

One sequence, two ribozymes: implications for the emergence of new ribozyme folds.

We describe a single RNA sequence that can assume either of two ribozyme folds and catalyze the two respective reactions. The two ribozyme folds share no evolutionary history and are completely different, with no base pairs (and probably no hydrogen bonds) in common. Minor variants of this sequence are highly active for one or the other reaction, and can be accessed from prototype ribozymes through a series of neutral mutations. Thus, in the course of evolution, new RNA folds could arise from preexisting folds, without the need to carry inactive intermediate sequences. This raises the possibility that biological RNAs having no structural or functional similarity might share a common ancestry. Furthermore, functional and structural divergence might, in some cases, precede rather than follow gene duplication.

Structurally complex and highly active RNA ligases derived from random RNA sequences.

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.

Two small temporal RNAs (stRNAs), lin-4 and let-7, control developmental timing in Caenorhabditis elegans. We find that these two regulatory RNAs are members of a large class of 21- to 24-nucleotide noncoding RNAs, called microRNAs (miRNAs). We report on 55 previously unknown miRNAs in C. elegans. The miRNAs have diverse expression patterns during development: a let-7 paralog is temporally coexpressed with let-7; miRNAs encoded in a single genomic cluster are coexpressed during embryogenesis; and still other miRNAs are expressed constitutively throughout development. Potential orthologs of several of these miRNA genes were identified in Drosophila and human genomes. The abundance of these tiny RNAs, their expression patterns, and their evolutionary conservation imply that, as a class, miRNAs have broad regulatory functions in animals.

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.

Synthesis of substituted N-benzyl pyridones via an O- to N-alkyl migration.

A new LiI-promoted O- to N-alkyl migration has been developed for the conversion of O-alkylated 2-hydroxy pyridines, quinolines, and pyrimidines to the corresponding N-alkylated heterocycles in good to excellent yields (57-99%). This method serves as an efficient means for the preparation of N-benzyl pyridones, quinolones, and pyrimidones.

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.

Identification by site-directed mutagenesis of Lys-558 as the covalent attachment site of H2DIDS in the mouse erythroid band 3 protein.

After functional expression of mouse erythroid band 3 by cRNA microinjection into Xenopus oocytes, 36Cl- efflux is irreversibly inhibited by H2DIDS. When a cRNA is injected that is derived from a cDNA in which the nucleotides encoding for lysine-558 were replaced by nucleotides encoding for asparagine, transport and inhibition of transport by H2DIDS still occur. However, when measured under conditions where no intramolecular crosslinking takes place the inhibition by H2DIDS is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 takes place at Lys-558.

pH-dependence of inhibition by H2DIDS of mouse erythroid band 3-mediated Cl- transport in Xenopus oocytes. The effect of oligonucleotide-directed replacement of Lys-558 by an Asn residue.

The rapid reversible inhibition of band 3-mediated inorganic anion transport by 4,4'-diisothiocyanodihydrostilbene-2,2'-disfulfonate (H2DIDS) turns slowly into irreversible inhibition. This is due to covalent bond formation of the two isothiocyanate groups of the inhibitor with two lysine residues on band 3, called Lys a and Lys b. In the red cell membrane, the pK value of Lys a is about 2.5 pK units lower than the pK value of Lys b. Hence the susceptibility of Lys a to irreversible modification by H2DIDS far exceeds the susceptibility to Lys b. In the present paper, we have expressed in Xenopus oocytes cRNA's derived from cDNA clones encoding wild-type mouse band 3 and mouse band 3 in which Lys a (Lys-558) had been replaced by an Asn residue by oligonucleotide-directed mutagenesis. In accord with previous findings, in the oocytes both wild-type and mutated band 3 mediate Cl- exchange. After determining the uninhibited exchange rate the oocytes were exposed for a fixed length of time to H2DIDS at a concentration (20 microM) which saturates all H2DIDS binding sites with reversibly bound H2DIDS (KI = 0.3 microM and 1.1 microM, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H2DIDS was replaced by bovine serum albumin to remove free and reversibly bound H2DIDS from the extracellular phase. Subsequent measurements of Cl- efflux yielded a measure for the irreversible inhibition that persisted. Since the transition from reversible to irreversible H2DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, pK values were calculated. These calculations could be made since in the wild-type, in which Lys a and Lys b are present, the exposure to H2DIDS could be confined to a pH range in which little if any covalent binding to Lys b takes place. The data could be represented by a single pK value of 8.3. In the mutant, Lys a is missing. Hence, covalent reaction can only take place with Lys b. Measurements over the appropriate pH range could be described by a single pK of 10.8. These values are 0.8-0.9 pK units higher than those previously obtained in experiments with band 3 in the red cell membrane (Kampmann et al. (1982) J. Membr. Biol. 70, 199-216).(ABSTRACT TRUNCATED AT 250 WORDS)

Accessing rare activities from random RNA sequences: the importance of the length of molecules in the starting pool.

BACKGROUND: In the past few years numerous binding and catalytic motifs have been isolated from pools of random nucleic acid sequences. To extend the utility of this approach it is important to learn how to design random-sequence pools that provide maximal access to rare activities. In an effort to better define the relative merits of longer and shorter pools (i.e. pools with longer or shorter random-sequence segments), we have examined the inhibitory effect of excess arbitrary sequence on ribozyme activity and have evaluated whether this inhibition overshadows the calculated advantage of longer pools. RESULTS: The calculated advantage of longer sequences was highly dependent on the size and complexity of the desired motif. Small, simple motifs were not much more abundant in longer molecules. In contrast, larger motifs, particularly the most complex (highly modular) motifs, were much more likely to be present in longer molecules. The experimentally determined inhibition of activity by excess sequence was moderate, with bulk effects among four libraries ranging from no effect to 18-fold inhibition. The median effect among 60 clones was fivefold inhibition. CONCLUSIONS: For accessing simple motifs (e.g. motifs at least as small and simple as the hammerhead ribozyme motif), longer pools have little if any advantage. For more complex motifs, the inhibitory effect of excess sequence does not approach the calculated advantage of pools of longer molecules. Thus, when seeking to access rare activities, the length of typical random-sequence pools (< or = 70 random positions) is shorter than optimal. As this conclusion holds over a range of incubation conditions, it may also be relevant when considering the emergence of new functional motifs during early evolution.

Selection and design of high-affinity RNA ligands for HIV-1 Rev.

We have used in vitro selection to isolate minimal, high-affinity RNA ligands for the Rev protein of HIV-1. Sequence analysis reveals that the tightest binding aptamers exhibit some similarity to a Rev-binding element (RBE) localized within the Rev-responsive element (RRE), but also contain novel sequence and structural motifs. A short helical stem and bulged nucleotides (nt) CUC ... UYGAG that have no counterpart in the wild-type (wt) element contribute to high-affinity binding. We have designed and synthesized a short (37 nt) RNA molecule that incorporates this motif; this RNA ligand has from three- to fivefold tighter binding than the full-length wt element, and up to 16-fold tighter than minimal wt RBEs. A guanosine:guanosine pairing that is postulated to occur in the wt element has been altered to other base pairings in the context of our optimized minimal element. RNAs that contain non-Watson-Crick base pairings, that can be modeled as isosteric to the wt G:G pair, bind Rev up to 160-fold tighter than elements that contain canonical Watson-Crick pairings or non-isosteric mismatches. These results support the hypothesis that Rev recognizes structural features associated with a non-Watson-Crick base pair.

The diagnosis and classification of multiple sclerosis: evoked responses and spinal fluid electrophoresis.

Multimodality evoked responses (including visual, brainstem auditory, and somatosensory) and CSF analysis were evaluated in 123 patients grouped into definite, probable, and possible MS according to the McAlpine criteria. The evoked responses (ERs) were very sensitive in detecting asymptomatic lesions and can therefore be used in conjunction with clinical data to provide evidence of multiple lesions. The CSF abnormalities also have high sensitivity and specificity in MS. ER and CSF findings, therefore, should be considered in addition to the clinical data in any classification of MS.

Working with families of suddenly and critically ill children: physician experiences.

OBJECTIVE: To describe physicians' experiences in attempting to provide optimal care for families of children who suffer from sudden, acute life-threatening conditions (SALTC). DESIGN: To generate descriptive data in this exploratory study, we used qualitative methods including focus groups and in-depth interviews. Transcripts of focus groups and interviews were analyzed for content using standard phenomenologic analysis methods, which resulted in a participant-generated conceptual model of optimal care for families of children with SALTC. SETTING: The intensive care unit of an urban pediatric teaching hospital. PARTICIPANTS: Twenty-two pediatric intensive care unit physicians, including residents, fellows, and attendings. INTERVENTION: None. MAIN OUTCOME MEASURES: Each participating physician provided qualitative descriptions of experiences caring for families of children with SALTC. RESULTS: Physicians identified 4 components of optimal care for families: (1) providing timely, accurate information about their child; (2) maintaining privacy for confidential discussions and personal grieving; (3) giving adequate emotional support; and (4) granting family members the right to hold and comfort their dying child. Physicians also described barriers to, and facilitators of this optimal care. CONCLUSIONS: Descriptive information provided in this exploratory study offers a complex model of optimal family care. Issues that affect the quality of care to families include those related to the context of providing care in a large teaching hospital, as well as subtleties of communication between parents and staff. Physicians' beliefs about optimal care of families in the pediatric intensive care unit revealed implications for both practice and training in pediatrics.

Load transfer with the Austin Moore cementless hip prosthesis.

More than 1,300 Austin Moore hemiarthroplasties have been reviewed in the literature, with no reports of fracture of the stem. Many patients with these hip implants had good function. The lack of stem fractures in patients with good functions has not been explained and contrasts with stem fractures that have occurred in patients with cemented prostheses of other designs during the same time. We used three-dimensional finite-element analysis and free-body diagrams to explain the lack of fractures for this device by a description of the probable load-transfer mechanisms between the prosthesis and the bone. Results from our finite-element analysis indicate that, with good calcar-collar support, the stresses in the stem are small because the stem portion of the prosthesis and the bone are uncoupled and, consequently, do not share the resultant bending moment of the head and abductor forces. If the stem is coupled to the bone so that the resultant bending moment is shared, high stresses in the stem are predicted; such stresses are inconsistent with the complete absence of fractures of these prostheses. The results of the finite-element analysis further showed that loss of calcar-collar support with proximal fixation through the fenestrations resulted in high stresses in the stem and stress shielding of the proximal medial cortex. The uncoupled prosthesis also may be modeled with a free-body diagram as a three-force member loaded at the head, stem tip, and in the proximal region. With this model, it can be shown that the reaction force of the stem tip, and thus the peak bending stress in the stem, increases as calcar-collar support is decreased. If there is no calcar-collar support, proximal support must be provided by some combination of integration of bone in the fenestrations and wedging due to the lateral-medial taper of the device. Stresses in the stem are largest when there is no wedging, but high stresses develop in the cancellous bone in the fenestrations. When there is wedging, stresses in the stem can be low, but stresses in the supporting cancellous bone can be high; additional proximal support through the fenestrations substantially reduces these bone stresses. If reduced stresses in the cancellous bone are indicative of a stable device, these mechanisms indicate that fractures of the Austin Moore prosthesis have not occurred in normally loaded hips because load was transferred primarily either through the collar or by wedging, with additional support at the fenestrations.

Influence of stem geometry on mechanics of cemented femoral hip components with a proximal bond.

Nonlinear, three-dimensional, finite element models of cemented femoral hip components with a proximal stem-cement bond were developed with use of a Charnley stem geometry and a modified Charnley stem geometry that had a cylindrical cross section over the distal two-thirds of the stem (Distal-Round). Peak tensile stresses in the proximal cement mantle increased 63 and 74% for the Charnley and Distal-Round stems, respectively, when the proximal stem-cement interface was debonded. The shear stresses over the stem-cement interface with a proximal bond were 29% larger for the Distal-Round stem than for the Charnley stem. After the proximal stem-cement interface was debonded, the peak tensile stresses in the cement mantle were 15% larger for the Distal-Round stem than for the Charnley stem. The results illustrate that stresses within the proximal cement mantle could be substantially reduced for both Charnley and Distal-Round stems through use of a proximal stem-cement bond. However, the risk of debonding may be higher for the Distal-Round stem because of increased shear stresses, and once debonded the risk of further loosening due to failure of the cement mantle would also be higher for the Distal-Round stem.

Residual stresses in ultra-high molecular weight polyethylene loaded cyclically by a rigid moving indenter in nonconforming geometries.

The characterization of stress and deformation fields that incorporate moving cyclic loads and nonlinear material response in ultra-high molecular weight polyethylene components for total knee replacements is required to quantify mechanisms of surface damage. A simulation of stresses in polyethylene components for total knee replacement subjected to cyclic moving loads was performed with use of nonlinear finite element analysis. Convergence to a steady-state cycle of stress and deformation was observed within five cycles of loading. Differential plastic deformation under the surface of the polyethylene led to horizontal residual stresses that were tensile at the surface and compressive in the subsurface. The magnitudes of the residual stresses indicate their importance in surface failure mechanisms. Horizontal residual tensile stresses at the surface are consistent with the initiation and propagation of surface cracks that could cause pitting in polyethylene. Horizontal residual compressive stresses under the surface could cause such cracks to arrest or turn and thus limit damage to a region just beneath the surface. The results emphasize the importance of incorporating nonlinear effects to simulate long-term stress fields associated with surface damage in polyethylene.

Degradation rate of ultra-high molecular weight polyethylene.

Ultra-high molecular weight polyethylene components for total joint replacement chemically degrade before and after implantation, and the degradation is associated with an increase in density. The goal of this study was to determine the average rate of density change in these components following sterilization by gamma radiation in air as a function of shelf age and implantation time. Using the density gradient column method, density profiles were obtained through the thickness from loaded and unloaded regions of 10 retrieved Insall-Burstein/Posterior-Stabilized II tibial components and one operating-room inventory component for which the initial density profile and patient history (if applicable) were known. The average density of the components increased at a constant rate of 0.000186 g/cc/month during the first 50 months after sterilization (r2 = 0.54) but was not significantly affected by loading (p > 0.05). The quantitative degradation rates may be useful to help verify kinetic models to predict bulk degradative changes on the basis of micro-structural and chemical processes. This research also suggests the hypothesis that degradation of ultra-high molecular weight polyethylene can be modeled in terms of changes in bulk or average properties.