[Prolonged weaning during early neurological and neurosurgical rehabilitation : S2k guideline published by the Weaning Committee of the German Neurorehabilitation Society (DGNR)]. / Prolongiertes Weaning in der neurologisch-neurochirurgischen Frührehabilitation : S2k-Leitlinie herausgegeben von der Weaning-Kommission der Deutschen Gesellschaft für Neurorehabilitation e. V. (DGNR).

Prolonged weaning of patients with neurological or neurosurgery disorders is associated with specific characteristics, which are taken into account by the German Society for Neurorehabilitation (DGNR) in its own guideline. The current S2k guideline of the German Society for Pneumology and Respiratory Medicine is referred to explicitly with regard to definitions (e.g., weaning and weaning failure), weaning categories, pathophysiology of weaning failure, and general weaning strategies. In early neurological and neurosurgery rehabilitation, patients with central of respiratory regulation disturbances (e.g., cerebral stem lesions), swallowing disturbances (neurogenic dysphagia), neuromuscular problems (e.g., critical illness polyneuropathy, Guillain-Barre syndrome, paraplegia, Myasthenia gravis) and/or cognitive disturbances (e.g., disturbed consciousness and vigilance disorders, severe communication disorders), whose care during the weaning of ventilation requires, in addition to intensive medical competence, neurological or neurosurgical and neurorehabilitation expertise. In Germany, this competence is present in centers of early neurological and neurosurgery rehabilitation, as a hospital treatment. The guideline is based on a systematic search of guideline databases and MEDLINE. Consensus was established by means of a nominal group process and Delphi procedure moderated by the Association of the Scientific Medical Societies in Germany (AWMF). In the present guideline of the DGNR, the special structural and substantive characteristics of early neurological and neurosurgery rehabilitation and existing studies on weaning in early rehabilitation facilities are examined.Addressees of the guideline are neurologists, neurosurgeons, anesthesiologists, palliative physicians, speech therapists, intensive care staff, ergotherapists, physiotherapists, and neuropsychologists. In addition, this guideline is intended to provide information to specialists for physical medicine and rehabilitation (PMR), pneumologists, internists, respiratory therapists, the German Medical Service of Health Insurance Funds (MDK) and the German Association of Health Insurance Funds (MDS). The main goal of this guideline is to convey the current knowledge on the subject of "Prolonged weaning in early neurological and neurosurgery rehabilitation".

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments.

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a beta-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.

Interdomain interactions within the gene 3 protein of filamentous phage.

Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p). The g3p consists of three domains (g3p-D1, D2 and D3) linked by flexible glycine-rich linkers. All three domains are indispensable for phage infectivity; the g3p-D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p-D2 domain to the F-pilus and the g3p-D3 domain anchors g3p to the phage coat. The N-terminal domains g3p-D1 and D2 interact with each other; this interaction is abrogated by binding of g3p-D2 to the F-pilus leading to the release of g3p-D1 to bind to TolA. Here, using phages with deletions in g3p, we have discovered a specific interaction between the two N-terminal domains and g3p-D3, the C-terminal domain of g3p. We propose that these interdomain interactions within g3p lead to a compact and stable organisation when displayed on the phage tip, but that during infection, this compact state must be unraveled.

Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 A: evidence for conformational lability.

Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F' pilus and the periplasmic protein TolA. Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F' pilus. In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2-217) from filamentous phage fd to 1.9 A resolution and compared it to the recently published structure of the same fragment from the related Ff phage M13. While the structure of individual domains D1 and D2 of the two phages are very similar (rms<0.7 A), there is comparatively poor agreement for the overall D1D2 structure (rms>1.2 A). This is due to an apparent movement of domain D2 with respect to D1, which results in a widening of the inter-domain groove compared to the structure of the homologous M13 protein. The movement of D2 can be described as a rigid-body rotation around a hinge located at the end of a short anti-parallel beta-sheet connecting domains D1 and D2. Structural flexibility of at least parts of the D1D2 structure was also suggested by studying the thermal unfolding of g3p: the TolA binding site on D1, while fully blocked by D2 at 37 degrees C, becomes accessible after incubation at temperatures as low as 45 degrees C. Our results support a model for the early steps of phage infection whereby exposure of the coreceptor binding site on D1 is facilitated by a conformational change in the D1D2 structure, which in vivo is induced by binding to the F' pilus on the host cell and which can be mimicked in vitro by thermal unfolding.

Single domain antibodies: comparison of camel VH and camelised human VH domains.

The antigen binding sites of conventional antibodies are formed primarily by the hypervariable loops from both the heavy and the light chain variable domains. Functional antigen binding sites can however also be formed by heavy chain variable domains (VH) alone. In vivo, such binding sites have evolved in camels and camelids as part of antibodies, which consist only of two heavy chains and lack light chains. Analysis of the differences in amino acid sequence between the VHs of these camel heavy chain-only antibodies and VH domains from conventional human antibodies helped to design an altered human VH domain. This camelised VH proved, like the camel VH, to be a small, robust and efficient recognition unit formed by a single immunoglobulin (Ig) domain. Biochemical, structural and antigen binding characterisation properties of both camel VH domains and camelised human VH domains suggest that these can compete successfully with single chain variable domain (Fv) fragments from conventional antibodies in many applications. Of special importance in this respect is the use of such VH domains as enzyme inhibitors, for which they seem to be better suited than Fv fragments. This function appears to be closely related to their often very long third hypervariable loop, which is central for antigen recognition in their binding sites.

The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.

Filamentous bacteriophages infecting gram-negative bacteria display tropism for a variety of pilus structures. However, the obligatory coreceptor of phage infection, postulated from genetic studies, has remained elusive. Here we identify the C-terminal domain of the periplasmic protein TolA as the coreceptor for infection of Escherichia coli by phage fd and the N-terminal domain of the phage minor coat protein g3p as its cognate ligand. The neighboring g3p domain binds the primary receptor of phage infection, the F pilus, and blocks TolA binding in its absence. Contact with the pilus releases this blockage during infection. Our findings support a sequential two-way docking mechanism for phage infection, analogous to infection pathways proposed for a range of eukaryotic viruses including herpes simplex, adenoviruses, and also lentiviruses like HIV-1.

A conserved infection pathway for filamentous bacteriophages is suggested by the structure of the membrane penetration domain of the minor coat protein g3p from phage fd.

BACKGROUND: . Gene 3 protein (g3p), a minor coat protein from bacteriophage fd mediates infection of Escherichia coli bearing an F-pilus. Its N-terminal domain (g3p-D1) is essential for infection and mediates penetration of the phage into the host cytoplasm presumbly through interaction with the Tol complex in the E. coli membranes. Structural knowledge of g3p-D1 is both important for a molecular understanding of phage infection and of biotechnological relevance, as g3p-D1 represents the primary fusion partner in phage display technology. RESULTS: . The solution structure of g3p-D1 was determined by NMR spectroscopy. The principal structural element of g3p-D1 is formed by a six-stranded beta barrel topologically identical to a permutated SH3 domain but capped by an additional N-terminal alpha helix. The presence of structurally similar domains in the related E. coli phages, lke and 12-2, as well as in the cholera toxin transducing phage ctxφ is indicated. The structure of g3p-D1 resembles those of the recently described PTB and PDZ domains involved in eukaryotic signal transduction. CONCLUSIONS: . The predicted presence of similar structures in membrane penetration domains from widely diverging filamentous phages suggests they share a conserved infection pathway. The widespread hydrogen-bond network within the beta barrel and N-terminal alpha helix in combination with two disulphide bridges renders g3p-D1 a highly stable domain, which may be important for keeping phage infective in harsh extracellular environments.

Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding.

BACKGROUND: Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors. OBJECTIVES: To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands. STUDY DESIGN: Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding. RESULTS: In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NIP VH) and from 3.1 microM to 1.6 microM (CDR2 randomisation of an anti-lysozyme VH). CONCLUSIONS: Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations of affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability.

Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.2 degrees C of the original human VH. Its stability was improved (melting point 61.6 degrees C) through three mutations to mimic camelid VHs even further: Va137 was replaced by phenylalanine and two cysteines were introduced at position 33 and 100b. The resulting VH folded properly and formed a second intradomain disulphide between the extra cysteines. The new mutations were then built constitutively into a phage-display VH library, from which antigen-specific VHs were selected. Two were analysed for stability with melting points of 72.6 and 75.3 degrees C. Thus secondary camelization enabled the isolation of VHs with improved folding stabilities exceeding even that of the original human VH. This indicates an effect on folding stability for some mutations specific in the light chain lacking camelid heavy chains.

Rearrangement of the former VL interface in the solution structure of a camelised, single antibody VH domain.

The solution structure of the isolated antibody heavy chain variable domain (VH)-P8 was determined by NMR spectroscopy. The VH had previously been modified (camelised) at three positions in its former antibody light chain variable domain (VL) interface to reduce hydrophobicity by mimicking camelid heavy chains naturally devoid of light chains. The architecture of two pleated beta-sheets and the conformation of the H1 and H2 loops in VH-P8 are very similar to those in non-camelised, VL-associated VH domains. Major differences concern the H3 loop, which no longer points towards the now absent VL, and three residues in the former VL interface. The side-chains of Val37 and Trp103 are buried and the Arg38 side-chain exposed in VH-P8. In non-camelised, VL-associated VH domains the side-chains of Val37 and Trp103 are in contact with the VL while the Arg38 side-chain is buried within the VH. Reorientation of Trp103 is due to the local structure in the beta-bulge of strand G. Reorientation of Val37 and Arg38 is caused by a disruption of regular beta-structure in strand C opposite the beta-bulge in strand C'. These changes, combined with the more hydrophilic side-chains of the camelised residues, reduce hydrophobicity and prevent non-specific binding of camelised VH domains, which proved critical for their use as small recognition units. The VH-P8 structure also indicates structural reasons for two other mutations specific for light-chain-lacking camel immunoglobins. Absence of the VH-typical Arg94/Asp101 salt bridge at the base of the H3 loop in VH-P8 may explain why a positively charged residue at position 94 is not conserved in camels. Reorientation of Val37 suggests a function of the camel-specific phenylalanine residue at this position in the hydrophobic core of light-chain-lacking camel heavy chains.

An antibody VH domain with a lox-Cre site integrated into its coding region: bacterial recombination within a single polypeptide chain.

Bacterial lox-Cre recombination within a single antibody VH domain was achieved through integration of a loxP site into its coding sequence. The 5' half of the VH gene, in which the H2 loop was replaced by a mutant loxP site, was fused to geneIII in an 'acceptor' fd-phage vector containing also a wild type loxP site. With a 'donor' plasmid vector harbouring the 3' half of the VH gene flanked by the same, differing loxP sites it recombined into a full-length VH with the loxP site-H2 loop. This VH was purified from bacterial periplasm, where it folded into a typical immunoglobulin domain. The system allows the generation of large VH repertoires using lox-Cre recombination.

Backbone assignment, secondary structure and protein A binding of an isolated, human antibody VH domain.

Antibody heavy chain variable domains (VH) lacking their light chain (VL) partner are prime candidates for the design of minimum-size immunoreagents. To obtain structural information about isolated VH domains, a human VH was labelled with 15N or doubly labelled with both 15N and 13C and was studied by heteronuclear nuclear magnetic resonance spectroscopy. Most (90%) of the 1H and 15N main-chain signals were assigned through two-dimensional TOCSY and NOESY experiments on the unlabelled VH and three-dimensional heteronuclear multiple quantum correlation TOCSY and NOESY experiments on the 15N-labelled VH. Four short stretches of the polypeptide chain could only be assigned on the basis of three-dimensional HNCA and HN(CO)CA experiments on the 13C-/15N-labelled protein. Long-range interstrand backbone NOEs suggest the presence of two adjacent beta-sheets formed by altogether nine antiparallel beta-strands. 3JNHC alpha H coupling constants and the location of slowly exchanging backbone amides support this interpretation. The secondary structure of the isolated VH is identical to that of heavy chain variable domains in intact antibodies, where VH domains are packed against a VL domain. The backbone assignments of the VH made it possible to locate its Protein A binding site. Chemical shift movements after complexing with the IgG binding fragment of Protein A indicate binding through one of the two beta-sheets of the VH. This beta-sheet is solvent exposed in intact antibodies. The Protein A binding site obviously differs from that on the Fc portion of immunoglobulins and is unique to members of the human VHIII gene subgroup.

Antibody VH domains as small recognition units.

To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH. Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface. These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners. The third hypervariable loop of the modified VH was then randomised to yield a repertoire of 2 x 10(8) independent clones, which was displayed on phage and selected through antigen binding. VH clones specific for hapten and protein antigens were isolated. Soluble VH was expressed with an isoleucine residue at position 47 to improve expression and stability compared to VH containing a glycine residue at this position, which however was preferable for phage selection. Affinities of soluble VH for hapten were between 100 nM and 400 nM. The VH domains were highly specific, stable and well expressed in Escherichia coli. These positive biophysical properties and their small size make them attractive for biotechnological applications.

'Camelising' human antibody fragments: NMR studies on VH domains.

A human heavy chain variable domain (VH) was expressed in bacteria for structural analysis by NMR spectroscopy. NMR analysis was initially impossible due to the short transverse proton relaxation time of the VH, probably caused by aggregation through the exposed interface naturally in contact with the light chain. The relaxation time was improved to normal values when this interface was mutated to mimic heavy chains of camel antibodies naturally devoid of light chains and through the use of the detergent CHAPS. Assignment of NMR signals will now be possible after isotopic labeling. Implications for the design of VH domains as minimum size immunoreagents are outlined.

Phage display and selection of a site-directed randomized single-chain antibody Fv fragment for its affinity improvement.

The affinity of an antibody Fv fragment was improved by semirational design involving site-directed randomization and phage display. On the basis of the predicted model of an anti-2-phenyloxazol-5-one (phOx) antibody Fv fragment, into which the ligand was inserted with the help of nuclear Overhauser enhancement (NOE) data, residues close to the hapten were identified. Seven of these residues in the third hypervariable regions of light and heavy chains were randomized in polymerase chain reactions (PCR) using degenerate oligonucleotides. Resulting clones were expressed as single-chain Fv (scFV) fragments on the surface of filamentous phage and selected for binding to phOx-conjugated bovine serum albumin. Selected Fv fragments were analyzed for hapten affinity by fluorescence quenching, and several mutants with improved affinities were identified. Phage selection on the basis of binding was very successful when phage scFv mutants differed in affinity by at least a factor of 6. Smaller differences did not result in predominant selection of the best binder. Combination of the two point mutations most crucial for improved hapten binding decreased the dissociation constant of the Fv for phOx 11-14-fold. Hapten binding of the improved Fv was analyzed in NOE experiments.

Improving the antigen affinity of an antibody Fv-fragment by protein design.

The affinity of an antibody for its ligand 2-phenyloxazolone was improved by protein design. For the design two-dimensional nuclear magnetic resonance spectroscopy, protein engineering and molecular modelling were used in an interactive scheme. Initially the binding site was localized with the help of transferred nuclear Overhauser enhancement signals from two, site specifically assigned tyrosine side-chains in the complementarity-determining regions of the antibody to the ligand 4-glycyl-2-phenyloxazolone. On their basis the hapten was placed into a model of the Fv-fragment built according to the principles of canonical antibody structures. From the model, unfavourable contacts between hapten and an aspartyl side-chain in complementarity-determining region 3 of the heavy chain were predicted. Substitution of the aspartyl residue by alanine resulted in a threefold increase in affinity of the antibody Fv-fragment for two hapten derivatives when compared with the wild-type. Nuclear magnetic resonance analysis of the improved Fv-fragment revealed an interaction between the alpha-carbon proton of alanyl residue with the ligand, which was not seen for the aspartyl residue. This interaction is not entirely in accordance with the model, which predicts an interaction between the side-chain of this residue and the hapten. However, it shows that by combined use of nuclear magnetic resonance analysis and molecular modelling, a residue that is critical for antigen binding was identified, whose mutation allowed the design of an improved antibody combining site.

Uniform labeling of a recombinant antibody Fv-fragment with 15N and 13C for heteronuclear NMR spectroscopy.

The expression of functional antibody fragments in Escherichia coli enables a detailed analysis by NMR spectroscopy. This is demonstrated with the uniform labeling of an Fv-fragment (25 kDa) comprising the antigen binding site of an antibody against 2-phenyloxazolone with 15N and 13C. The antigen-complexed Fv-fragment was analysed for a potential assignment by heteronuclear multi-dimensional NMR spectroscopy. For almost all backbone amides 15N/1H crosspeaks and for 80% of them TOCSY crosspeaks were observed. In a 13C-edited-HCCH-2D experiment 17 out of 18 threonine spin-systems were identified. Thus detailed assignments are possible, but some amino acid specific labeling in addition to uniform labeling will be required for complete assignments of Fv-fragments.

Use of 2D NMR, protein engineering, and molecular modeling to study the hapten-binding site of an antibody Fv fragment against 2-phenyloxazolone.

Two-dimensional (2D) 1H NMR spectroscopy was used to study the hapten-binding site of a recombinant antibody Fv fragment expressed in Escherichia coli. Point mutations of residues in the CDR loops of the Fv fragment were designed in order to investigate their influence on hapten binding and to make site-specific assignments of aromatic NMR proton signals. Two tyrosines giving NOEs to the ligand 2-phenyloxazolone were identified, residue 33 in CDR1 of the heavy chain and residue 32 in CDR1 of the light chain. The benzyl portion of 2-phenyloxazolone is located between these two residues. The binding site is close to the surface of the Fv fragment. Comparison with a different anti-2-phenyloxazolone antibody, the crystal structure of which has recently been solved, shows that the general location of the hapten-binding site in both antibodies is similar. However, in the crystallographically solved antibody, the hapten is bound farther from the surface in a pocket created by a short CDR3 loop of the heavy chain. In the binding site identified in the Fv fragment studied in this report, this space is probably filled by the extra seven residues of the CDR3.

Antigen-antibody interactions: an NMR approach.

With recent advances in methodology, it now appears that NMR can be used at an unprecedented level of sophistication to obtain new insights into the solution structure and dynamics of the antibody combining site, both free and in its complex with antigen. Most promising in this regard is the Fv fragment (molecular weight approximately 25 kD) which can be produced by genetic engineering in a form suitable for NMR studies. Isotopic labeling is required to make specific resonance assignments. NMR can also provide information on the conformational preferences of immunogenic peptides and can be used to probe the conformation and dynamics of peptides (appropriately labeled with 13C or 15N) bound to the Fab fragment (molecular weight approximately 50 kD) of antipeptide antibodies.