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Biomedical subjects

A Lahm

Publications and source records attributed to A Lahm.

At least 37 records · Page 2Linked to original sources
PubMed1999

Postoperative ossifications of the shoulder. Incidence and clinical impact.

Periarticular ossifications of the shoulder after surgery have been described since the beginning of the century. Risk factors and the clinical impact of heterotopic bone formation have been discussed controversially. After open surgery on the shoulder, 131 patients (rotator cuff repair n = 106, acromioplasty n = 25) were included in a retrospective study if pre- and postoperative X-rays were available. The age of the 90 men and 41 women averaged 51 years (range 29-67 years). The minimum follow-up was 2 years. Also, 108 patients were interviewed by questionnaire to estimate the subjective outcome of the procedure (5 patients were reported dead). A clinical examination was carried out on 86 patients using the Constant score for evaluation of the objective outcome. Heterotopic ossifications were found in 35 cases (26.7%), 28 of them after rotator cuff reconstruction and 7 after acromioplasty. A good to excellent result was reported by 89% (n = 65) of the patients without and by 80% (n = 28) of the patients with ossifications. The Constant score averaged 69 points and 74 points (n = 60), respectively. A significant difference between the two collectives could not be calculated. As significant risk factors for the formation of heterotopic bone, the existence of osteoarthritis and the duration and complexity of the procedure could be cited. The appearance of periarticular ossifications after surgery of the shoulder seems to be of minor clinical impact. Severe cases with major functional deficits should and can be prevented by a fast and atraumatic operation technique.

Acromion↗
PubMed1998

Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione.

Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.

Alanine↗
PubMed1998

Recognition of single-stranded DNA by nuclease P1: high resolution crystal structures of complexes with substrate analogs.

The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 A structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspergillus oryzae are discussed.

Catalysis↗
PubMed1998

Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.

Amino Acid Sequence↗
PubMed1998

Transglutaminase 1 mutations in lamellar ichthyosis. Loss of activity due to failure of activation by proteolytic processing.

Lamellar ichthyosis is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. It is caused by mutations in the TGM1 gene that encodes the transglutaminase 1 (TGase 1) enzyme, which is critical for the assembly of the cornified cell envelope in terminally differentiating keratinocytes. TGase 1 is a complex enzyme existing as both cytosolic and membrane-bound forms. Moreover, TGase 1 is proteolytically processed, and the major functionally active form consists of a membrane-bound 67/33/10-kDa complex with a myristoylated and palmitoylated amino-terminal 10-kDa membrane anchorage fragment. To understand better how point mutations, deletions, and truncations found in lamellar ichthyosis disease affect the structure and function of TGase 1, we have expressed in baculovirus and keratinocytes a number of reported TGase 1 mutants. The structural implications of these mutations were examined using a homology-derived three-dimensional model of TGase 1 generated from the known x-ray structure of the related coagulation factor XIIIa enzyme. The present studies demonstrate that loss of TGase 1 activity is not restricted to mutations that directly affect the enzymatic activity. We report a new class of mutations that impair the subsequent post-synthetic processing of the protein into its highly active functional forms.

Amino Acid Sequence↗
PubMed1998

Ankle joint arthroscopy for meniscoid lesions in athletes.

The meniscoid lesion is a frequent but not well known cause of persistent pain in the anterior part of the upper ankle in sports traumatology. It has been described as portions of hyalinized tissue following an inversion sprain of the ankle. Trapping of this formation between the lateral cheek of the talus and the fibula is supposed to be responsible for pain and other symptoms reported by the patient. In 59 arthroscopic procedures on the ankle joint in athletes, meniscoid lesions were seen in 19 cases. Only 1 of these 19 patients showed lateral and anterior instability, and frequent clinical symptoms were swelling and trapping. Intraoperatively, all meniscoid lesions were combined with synovitis. Chondromalacia and osteophytes were seen several times. After an average follow-up period of 12 months, 14 patients could be examined. Twelve of the athletes returned to full sports activity; 10 were very satisfied, 2 satisfied, and 2 unsatisfied. Nine patients did not complain of any swelling, 4 did so on rare occasions, and 1 complained persistently. No pain was reported 10 times, improvement of pain 3 times, and continuing persistent pain 1 time, probably because of simultaneous chondromalacia and osteophytes. These were found more frequently in patients with a longer case history and unsuccessful conservative treatment, so that early arthroscopic surgery is recommended.

Adult↗
PubMed1998

Articular and osseous lesions in recent ligament tears: arthroscopic changes compared with magnetic resonance imaging findings.

The treatment of ligament injuries of the knee has undergone rapid progress, especially with the improvement of arthroscopic reconstruction of the anterior cruciate ligament (ACL). Since the advent of magnetic resonance imaging (MRI) after knee trauma with ligament injuries, interest has focussed on the clinical significance of concomitant articular and osseous lesions. In 48 of 141 MRIs, different types of these lesions were found; in 38 cases an arthroscopy was performed and 34 times the patients could clinically and radiologically be examined after at least 6 months. Bone bruise was found 26 times, in 16 cases associated with ACL-tears. Eleven patients had subchondral fractures, 7 osteochondral fractures, and in 4 patients, stress fractures were found. They were attributed to various mechanisms of trauma, in different percentages associated with ligament tears and in different dimensions visible or progressive on follow-up MRIs. Obviously some of the different lesions of subchondral and spongeous bone can indicate later degenerative arthritis, so that we find hints for a modification of rehabilitation, e.g., open versus closed kinetic chain or orthosis with relief of single compartments.

Adolescent↗
PubMed1998

[Arthroscopic therapy of osteochondrosis dissecans of the talus--follow-up with a new "Ankle Score"].

INTRODUCTION: Since the advent of operative ankle arthroscopy specific treatment of osteochondritis dissecans of the talus underwent rapid progress. Besides optimizing well-known methods as drilling, spongeous plastic, curettage or refixation of dissecates new trends go to transplantation of cultivated cartilage and osteochondral allografts. Previous follow-up examinations suffer on the one hand from partially small numbers of cases, on the other hand comparisons are difficult because so far no rating system of the function of the upper ankle does exist. MATERIAL AND METHODS: Within three years 34 patients underwent arthroscopic treatment of osteochondritis dissecans of the talus, 16 with percutaneous drilling, 12 spongeous plastics, three refixations and three curettages. The average age of the 22 men and 12 women was 25 years (11-48 years). A newly developed score system and a follow up MRI was used in a follow-up of 29 of the patients. Up to 100 points are given in the categories pain, stability/insecurity, efficiency/painfree walking distance, gait, differences in circumference, motility and power. RESULTS: 27 of the 34 patients had a trauma history. 20 lesions were localized at the lateral talus, they all had a trauma history. In 7 of the 14 lesions at the medial part of the talus there was no evidence of trauma. The 29 patients in the follow-up achieved an average of 87 points, the 16 patients after K-wire drilling 85 points and the 12 patients after spongeous plastic 90 points. Deductions were noted likewise in the subjective and objective parameters. 100 points were reached by 4 patients. DISCUSSION: Cultured chondrozytes and osteochondral grafts are new trends in treatment of osteochondritis dissecans while arthroscopically controlled spongeous plastic after curettage and K-wire drilling represent the main component of early stages with intact or partially fractured cartilage surface. Results of K-wire drilling are negligible worse than those of spongeous plastic, which is attributed to a generous perforation of the sclerosis. This is contributed to an improved preoperative diagnosis with MRI.

Adolescent↗
PubMed1997

Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies.

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level.

Animals↗
PubMed1996

Engineering human interleukin-6 to obtain variants with strongly enhanced bioactivity.

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL-6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL-6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL-6Ralpha), potentiate IL-6 bioactivity and induce a cytokine-responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL-6Ralpha (shIL-6Ralpha) in sera, suggests that the hIL-6-shIL-6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL-6 (hIL-6) variants with strongly enhanced shIL-6Ralpha binding activity and bioactivity. Homology modeling and site-directed mutagenesis of hIL-6 suggested that the binding interface for hIL-6Ralpha is constituted by the C-terminal portion of the D-helix and residues contained in the AB loop. Four libraries of hIL-6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL-6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in shIL-6Ralpha binding; a further increase (up to 70-fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL-6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL-6Ralpha at concentrations similar to those normally found in human sera.

Antigens, CD↗
PubMed1996

Human interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells.

Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super-antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.

Antigens, CD↗
PubMed1996

[Revision surgery of knee endoprosthesis].

Thirty-one revisions for aseptic or septic loosening of knee arthroplasty have been performed between 1983 and 1995. In 18 cases we had loosening of uni- or bicondylar prosthesis and in 13 cases a tricompartmental revision arthroplasty. With an average of 53 months (1.5 to 13 years) after the last operation 21 patients could be examined. Main reasons for failure of uni- and bicondylar prosthesis were as well a proceeding of the arthritis in other compartments, instability, incorrect alignment and other reasons depending on the surgical technique. We found similar reasons in aseptic loosening of total knee arthroplasties including wrong choice of non-constrained condylar prosthesis. Seven cases of late infection affected semi-constrained prosthesis. Two of the reimplantations in a 2-stage procedure failed. Using the Insall-Score in the follow-up the patients reached 71.9 points in the knee score and 58.9 points in the functional score. Patients with former aseptic loosening reached better results than these with septic loosening. 38% were absolutely painfree, 14% complained about permanent pain. Unlimited walking was found 5 times, none of the examined persons was unable to walk. Main problems in revision surgery concern reconstruction of a good alignment and the management of bone loss.

Aged↗
PubMed1995

Definition of a composite binding site for gp130 in human interleukin-6.

The helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6R alpha subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6 IL-6R alpha subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitro and that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor.

Antigens, CD↗
PubMed1995

Interleukin-6 (IL-6) antagonism by soluble IL-6 receptor alpha mutated in the predicted gp130-binding interface.

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal-transducing beta chain gp130. Since the cytoplasmic region of IL-6R alpha is not required for signal transduction, soluble forms of IL-6R alpha (sIL-6R alpha) show agonistic properties because they are still able to originate IL-6.sIL-6R alpha complexes, which in turn associate with gp130. A three-dimensional model of the human IL-6.IL-6R alpha.gp130 complex has been constructed and verified by site-directed mutagenesis of regions in shIL-6R alpha (where "h" is human) anticipated to contact hgp130, with the final goal of generating receptor variants with antagonistic properties. In good agreement with our structural model, substitutions at Asn-230, His-280, and Asp-281 selectively impaired the capability of shIL-6R alpha to associate with hgp130 both in vitro and on the cell surface, without affecting its affinity for hIL-6. Moreover, the multiple substitution mutant A228D/N230D/H280S/D281V expressed as a soluble protein partially antagonized hIL-6 bioactivity on hepatoma cells.

Amino Acid Sequence↗
PubMed1995

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.

Amino Acid Sequence↗
PubMed1994

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin-6 (IL-6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post-menopausal osteoporosis. IL-6, a four-helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low-affinity IL-6 receptor (IL-6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL-6R alpha. These mutants behave as full and selective IL-6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL-6 super-antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL-6R alpha. The IL-6 super-antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild-type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL-6 suggests that it could be possible to design super-antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

Carcinoma, Hepatocellular↗