Biomedical subjects

S Freitag

Publications and source records attributed to S Freitag.

At least 19 recordsLinked to original sources

PubMed2006

Prevention of device-related tissue damage during percutaneous deployment of tissue-engineered heart valves.

BACKGROUND: Endovascular application of pulmonary heart valves has been recently introduced clinically. A tissue-engineering approach was pursued to overcome the current limitations of bovine jugular vein valves (degeneration and limited longevity). However, deployment of the delicate tissue-engineered valves resulted in severe tissue damage. Therefore the objective of this study was to prevent tissue damage during the folding and deployment maneuver. MATERIAL AND METHODS: Porcine pulmonary heart valves, small intestinal submucosa, and ovine carotid arteries were obtained from a slaughterhouse. After dissection and antimicrobial incubation, the valves were trimmed (removal of sinus and most of the muscular ring) to fit into the deployment catheter. The inside (in-stent group, n = 6) or outside (out-stent group, n = 6) of a nitinol stent was covered by an acellular small intestinal submucosa, and the valves were sutured into the stent. The valves were folded, tested for placement in the deployment catheter, and decellularized enzymatically. Myofibroblasts were obtained from carotid artery segments and seeded onto the scaffolds. The seeded constructs were placed in a dynamic bioreactor system and cultured for 16 consecutive days. After endothelial cell seeding, the constructs were folded, deployed, and processed for histology and surface electron microscopy. RESULTS: The valves opened and closed competently throughout the entire dynamic culture. Surface electron microscopy revealed an almost completely preserved tissue in the in-stent group. Stents covered with small intestinal submucosa on the outside, however, showed severe damage. CONCLUSION: This study demonstrates that small intestinal submucosa covering of the inside of a pulmonary valved stent can prevent stent strut-related tissue damage.

Animals↗

PubMed2006

Aesthetic effect of minor changes in incisor angulation: an internet evaluation.

The aim of this study was to evaluate the aesthetic assessment of different tiltings of the upper incisors with surrounding tissues. A picture of a smiling mouth revealing the upper anterior teeth from 15 to 25 was digitally transformed producing different tiltings of the incisors. These seven versions presented in a web-based survey were evaluated concerning their attractiveness. Overall 439 judges joined the evaluation. However, 249 judges were included in analysis, because only judges with normal wellbeings and completed questionnaires were accepted. These judges consisted of 38 dentists, 26 dental and 24 other students, 45 academics and 89 non-academics, while 27 gave no data about their profession. The results [median (first; third quartile)] showed significant that symmetrical incisors [67.5 (47.5; 85.0)] and minor changes in the angulation of one [65.0 (42.5; 80.0)] or both lateral incisors [65.0 (42.5; 80.0)], respectively, are aesthetically more attractive than the angulation of one [45.0 (27.5; 62.5)] or both central incisors [27.5 (16.3; 45.0)], or the combination of one tilted lateral and central incisor [37.5 (25.0; 60.0)]. These results confirm the results of a previous study where similar image versions without surrounding tissues were assessed concerning their attractiveness on printed photos.

Beauty↗

PubMed2006

Subjective and objective perception of upper incisors.

The purpose of this study was to evaluate the subjective judgment (SJ) of patients on their own dental appearance and to correlate the results with objective measurements (OM) of their dentition concerning the appearance of the upper incisors. Seventy-five participants (30 men and 45 women) with normal well-being were included in the study. In a questionnaire they judged the appearance of their upper incisors. Furthermore, OM were evaluated by the investigator with regard to the following points: (i) absolute length of the upper central incisors, (ii) their length exposed during laughing, (iii) width-to-length ratio of central incisors and (iv) the proportion between the width of the lateral and central incisors. The subjective results were registered on visual-analogue scales. For the objective results standardized photographs were taken. No gender dependent differences could be found for the objectively measured parameters (median): OM1, 10.7 mm; OM2, 8.1 mm; OM3, 0.81; OM4, 0.79. However, significant correlations between subjective and objective results (SJ1/OM1, SJ2/OM2, SJ3/OM3) could be shown for men, but not for women. The maximum of the calculated regression-curves for men reflect 'golden standard values' well known from the literature. The degree of satisfaction concerning appearance of anterior incisors in accordance with golden standard values is higher for men than for women.

Adult↗

PubMed2005

Influence of metal cleaning methods on the resin bond strength to NiCr alloy.

OBJECTIVES: The purpose of this laboratory study was to evaluate the influence of different metal cleaning methods on the bond strength of various resin bonding systems and its durability to a NiCr alloy. The hypothesis was that the resin bond strength and its durability is related to the cleaning method and to the bonding system used. METHODS: Plexiglas tubes filled with composite resin were bonded to NiCr disks using three different bonding systems: tribochemical silica-coating and silanization of the alloy, acrylization of the alloy, or bonding with a phosphate monomer containing composite resin. For each bonding system, three cleaning methods were used after the last air abrasion step: cleaning with a stiff brush, ultrasonic cleaning in alcohol or in a neutral detergent. For each combination 20 samples were bonded. Subgroups of 10 bonded samples were tested for tensile strength following storage for either 3 or 150 days. In addition, the 150-day samples were thermal cycled 37,500 times. The statistical analyses were made by ANOVA, followed by multiple pair-wise comparison of the groups using Scheffe and t-tests with Bonferroni-Holm correction. RESULTS: The mean tensile bond strengths ranged from 34.8 to 49.2 MPa after 3 days and from 2.1 to 43.1 MPa after 150 days. Acrylization of the alloy did not provide durable bond strength to NiCr alloy over storage time. Cleaning the alloy surface with alcohol provided statistically significant higher bond strength than the other cleaning methods after 3 days. SIGNIFICANCE: Alloy cleaning methods after air abrasion have a significant influence on the resin bond strength.

2-Propanol↗

PubMed2005

Matrix metalloproteinase-9 in bronchiolitis obliterans syndrome after lung transplantation.

Bronchiolitis obliterans syndrome (BOS) is a severe complication after lung transplantation (LTX). In a retrospective cohort study 12 stable healthy recipients (non-BOS) and eight patients with BOS were enrolled after LTX and matrix metalloproteinases (MMP)-9, TIMP-1 and cell characteristics in bronchoalveolar lavage (BAL) samples (n = 145) were analysed. BALs from patients with BOS were further divided according to whether they were obtained before (pre-BOS) or after manifestation of BOS (BOS group). The MMP-9/TIMP-1 ratio was significantly increased in the BOS group compared with non-BOS or pre-BOS; furthermore, the ratio was negatively correlated with forced expiratory volume in one second. In zymography, the active form of MMP-9 was detected predominantly in the BOS group. In addition, zymography showed the banding pattern of neutrophil-derived MMP-9, indicating that polymorphonuclear neutrophils (PMNs) were the main source of MMP-9. According to that, MMP-9 was significantly correlated with the number of PMN. In immunocytochemistry, MMP-9 was also associated predominantly with PMN. This is the first study to evaluate the expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases-1 over time during manifestation of a fibroproliferative lung disease in patients. It demonstrates development of bronchiolitis obliterans syndrome after lung transplantation is associated with an imbalance of matrix metalloproteinases-9/tissue inhibitors of metalloproteinase-1 ratio.

Adult↗

PubMed2002

[Sensitivity of MRI in detecting alveolar Infiltrates: Experimental studies].

PURPOSE: An experimental study using porcine lung explants and a dedicated chest phantom to evaluate the signal intensity of artificial alveolar infiltrates with T 1 - and T 2 -weighted MRI sequences. MATERIAL AND METHODS: 10 porcine lung explants were intubated, transferred into the cavity of a MRI-compatible chest phantom and inflated by continuous evacuation of the artificial pleural space. All lungs were examined with MRI at 1.5 T before and after intra-tracheal instillation of either 100 or 200 ml gelatine-stabilised liquid to simulate alveolar infiltrates. MR-examination comprised gradient echo (2D- and 3D-GRE) and fast spin echo sequences (T 2 -TSE and T 2 -HASTE). The signal intensity of lung parenchyma was evaluated at representative cross sections using a standardised scheme. Control studies were acquired with helical CT. RESULTS: The instilled liquid caused patchy confluent alveolar infiltrates resembling the findings in patients with pneumonia or ARDS. CT revealed typical ground-glass opacities. Before the application of the liquid, only T 2 -HASTE and T 2 -TSE displayed lung parenchyma signals with a signal/noise ratio of 3.62 and 1.39, respectively. After application of the liquid, both T 2 -weighted sequences showed clearly visible infiltrates with an increase in signal intensity of approx. 30 % at 100 ml (p < 0.01) and 60 % at 200 ml (p < 0.01). With 2D- and 3D-GRE the infiltrates were not visible, although the lung parenchyma signal increase was statistically significant. On 2D-GRE the increase in signal intensity reached 0.74 % (p = 0.32) after 100 ml and 5.6 % (p < 0.01) after 200 ml (for 3D-GRE: 2.2 % [p = 0.02] at 100 ml and 4.4 % at 200 ml [p < 0.01]). The CT controls revealed a significant increase of lung density of 17 H.E. at 100 ml (p = 0.02) and 75 H.E. at 200 ml (p < 0.01). CONCLUSIONS: MRI with T 2 -weighted sequences detects artificial alveolar infiltrates with high signal intensity and may be a highly sensitive tool to detect pneumonia in patients.

Animals↗

PubMed2001

Ulcerative colitis and colorectal carcinoma: DNA-profile, laminin-5 gamma2 chain and cyclin A expression as early markers for risk assessment.

BACKGROUND: Ulcerative colitis patients are at increased risk for developing colorectal carcinomas. Despite expensive surveillance programmes, clinical practice reflects an uncertainty in individual risk assessment. The aim of the study was to evaluate independent cellular features with possible predictive value. METHODS: Two patient groups were selected: group A comprised 8 patients with ulcerative colitis-associated colorectal carcinomas, group B comprised 16 ulcerative colitis patients with risk factors (duration of disease, extent of inflammation, epithelial dysplasias). A total of 683 paraffin-embedded mucosal biopsies were retrospectively evaluated for inflammatory activity, grade of dysplasia, ploidy status, laminin-5 gamma2 chain and cyclin A expression. RESULTS: Mild or moderate inflammatory activity was present in 78% of all biopsies, low- or high-grade dysplasia in 5.5%. There was no difference in inflammatory activity and dysplasia between patient groups. In group A, 75% of the biopsies exhibited aneuploid DNA distribution patterns. Group B showed mainly proliferative-diploid cell populations (85% / P = 0.006). Laminin-5 gamma2 chain was expressed in 13% of all biopsies, with a higher frequency in group A (P = 0.002). Cyclin A expression was found in 98% of all biopsies, with a higher number of immunopositive cells in group A biopsies (P = 0.014). CONCLUSIONS: Combined nuclear DNA assessment, laminin-5 gamma2 chain and cyclin A expression may help to identify ulcerative colitis patients with an increased risk for cancer development.

Adult↗

PubMed2001

Jurkat cell-reactive anti-thymocyte globulin assessed ex vivo by flow cytometry persists three weeks in circulation.

Rabbit anti-thymocyte globulin (ATG-Fresenius) is a polyclonal anti-serum raised against the lymphoblastic T cell line Jurkat. It is used for in vivo depletion of host and donor T cells for allogeneic stem cell transplantation. After administration of 90 mg/kg prior to transplant, rabbit immunoglobulin G (IgG) remains present for 4-5 weeks, but it is unknown how long T cell-reactive antibodies persist. Therefore, we measured anti-Jurkat antibodies by flow cytometry. The detection limit for Jurkat-reactive antibodies was 0.1 microg/ml rabbit IgG; half-maximal labeling of Jurkat cells required 183 microg/ml rabbit ATG. The mean half-life of Jurkat-reactive antibodies in 7 patients was 4 days. Detectable levels persisted up to 3 weeks with antibody levels equivalent to 0.2-4.1 microg/ml rabbit ATG. Jurkat-reactive antibodies were eliminated two-fold faster than rabbit IgG, as assessed by enzyme-linked immunosorbent assay (ELISA). The results suggest that in patients pretreated with ATG before transplantation, residual anti T-cell antibodies may effectively modulate recovery of T cells generated after transplantation, thereby lowering the incidence of severe GVHD.

Animals↗

PubMed2000

Serovar specific risk factors and clinical features of Salmonella enterica ssp. enterica serovar Enteritidis: a study in South-West Germany.

Human salmonellosis in Germany has been an increasing problem since the 1980s, with a peak of 195,000 reported cases in 1992. During the peak years, isolates of Salmonella Enteritidis (SE) predominated by far over other salmonella serovars (NSE) (80 vs. 20%). In a comparison of the clinical characteristics of 790 persons infected with SE to 175 persons infected with NSE, watery diarrhoea (OR 1.7) and high grade (> 39 degrees C) fever (OR 1.8) were independently associated with SE infection. When comparing possible risk factors for acquiring salmonella infection among patients with SE compared to those with NSE, consumption of raw eggs (OR 4.4; P = 0.0006) was the most significant alimentary risk factor for SE infection, while travel outside Europe was negatively associated with SE infection (OR 0.08; P = 0.0001). When comparing all patients with salmonella infection, regardless of serovar, with healthy controls, consumption of raw eggs (OR 30.3; P = 0.001), of raw or undercooked eggs (OR 1.9; P = 0.003), or having puppies, kittens or turtles (OR 6.8; P = 0.002), were risk factors for salmonellosis.

Adolescent↗

PubMed2000

Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system.

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium deltaG degrees, deltaH degrees, deltaS degrees, deltaC degrees(p), and activation deltaG++ of S45A at 37 degrees C are 13.7+/-0.1 kcal/mol, -21.1+/-0.5 kcal/mol, -23.7+/-1.8 cal/mol K, -223+/-12 cal/mol K, and 20.0+/-2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the deltaH++ and deltaS++ of dissociation, 25.8+/-1.2 kcal/mol and 18.7+/-4.3 cal/mol K. The large increases of deltaH++ and deltaS++ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.

Binding Sites↗

PubMed1999

Streptavidin-biotin binding energetics.

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.

Binding Sites↗

PubMed1999

X-ray crystallographic studies of streptavidin mutants binding to biotin.

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.

Binding Sites↗

PubMed1999

A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway.

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.

Biotin↗

PubMed1999

Atomic resolution structure of biotin-free Tyr43Phe streptavidin: what is in the binding site?

The streptavidin-biotin system is an example of a high-affinity protein-ligand pair (Ka approximately 10(13) mol-1). The thermodynamic and structural properties have been extensively studied as a model system for protein-ligand interactions. Here, the X-ray crystal structure of a streptavidin mutant of a residue hydrogen bonding to biotin [Tyr43Phe (Y43F)] is reported at atomic resolution (1.14 A). The biotin-free structure was refined with anisotropic displacement parameters (SHELXL97 program package). The high-resolution data also allowed interpretation of side-chain and residue disorder in 41 residues where alternate conformations were refined. The Y43F mutation is unambiguously observed in difference maps, although only a single O atom per monomer is altered. The atomic resolution enabled the identification of 2-methyl-2, 4-pentanediol (MPD) molecules in the biotin-binding pocket for the first time. Electron density for MPD was observed in all four subunit binding sites of the tetrameric protein. This was not possible with data at lower resolution (1.8-2.3 A) for wild-type streptavidin or mutants in the same crystal form using MPD in the crystallization. The impact of MPD binding on these studies is discussed.

Binding Sites↗

PubMed1999

Click evoked myogenic potentials in the differential diagnosis of acute vertigo.

OBJECTIVE: In response to loud clicks, a vestibular evoked potential can be recorded from sternocleidomastoid muscles, called "click evoked myogenic potential" (CEMP). This paper reports on the usefulness of CEMP in the differential diagnosis of acute vertigo of presumed vestibular origin. METHODS: CEMP was examined in 40 patients with acute vertigo of vestibular origin (26 with acute peripheral vestibulopathy, five with Ménière's disease, three with benign paroxysmal positioning vertigo (BPPV), six with psychogenic vertigo) and the results compared with standard caloric reaction (CR). For CEMPs, clicks were delivered unilaterally via a pair of headphones. EMG activity was collected by surface electrodes placed on the sternocleidomastoid belly and averaged. RESULTS: In 29 patients, CR was unilaterally abnormal, pointing to a peripheral vestibular lesion. Seventeen of them had a corresponding loss of CEMPs; the other 12 patients had a normal CEMP. The remaining 11 patients had normal results in both tests. In comparison with CR, CEMP showed a sensitivity of 59% and a specificity of 100% for peripheral vestibular disorders. CONCLUSION: CR is a test of the horizontal canal whereas CEMP is thought to be a sacculus test. Different results of CR and CEMP may be due to this difference between target organs stimulated and may be of prognostic value.

Acoustic Stimulation↗

PubMed1998

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex.

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.

Binding Sites↗

PubMed1998

Biodiversity assessment and conservation strategies

The efficient representation of all species in conservation planning is problematic. Often, species distribution is assessed by dividing the land into a grid; complementary sets of grids, in which each taxon is represented at least once, are then sought. To determine if this approach provides useful surrogate information, species and higher taxon data for South African plants and animals were analyzed. Complementary species sets did not coincide and overlapped little with higher taxon sets. Survey extent and taxonomic knowledge did not affect this overlap. Thus, the assumptions of surrogacy, on which so much conservation planning is based, are not supported.

Journal Article↗

PubMed1998

Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system.

A circularly permuted streptavidin (CP51/46) has been designed to remove the flexible polypeptide loop that undergoes an open to closed conformational change when biotin is bound. The original termini have been joined by a tetrapeptide linker, and four loop residues have been removed, resulting in the creation of new N- and C-termini. Isothermal titration calorimetric studies show that the association constant has been reduced approximately six orders of magnitude below that of wild-type streptavidin to 10(7) M(-1). The deltaH degrees of biotin association for CP51/46 is reduced by 11.1 kcal/mol. Crystal structures of CP51/46 and its biotin complex show no significant alterations in the binding site upon removal of the loop. A hydrogen bond between Ser45 and Ser52 found in the absence of biotin is broken in the closed conformation as the side-chain hydroxyl of Ser45 moves to hydrogen bond to a ureido nitrogen of biotin. This is true in both the wild-type and CP51/46 forms of the protein, and the hydrogen bonding interaction might thus help nucleate closure of the loop. The reduced entropic cost of binding biotin to CP51/46 is consistent with the removal of this loop and a reduction in entropic costs associated with loop closure and immobilization. The reduced enthalpic contribution to the free energy of binding is not readily explainable in terms of the molecular structure, as the binding contacts are nearly entirely conserved, and only small differences in solvent accessible surfaces are observed relative to wild-type streptavidin.

Binding Sites↗