A novel approach for the homogenization of cellulose to use micro-amounts for stable isotope analyses.

Climate reconstructions using stable isotopes from tree-rings are steadily increasing. The investigations concentrate mostly on cellulose due to its high stability. In recent years the available amount of cellulose has steadily decreased, mainly because micro-structures of plant material have had to be analyzed. Today, the amounts of cellulose being studied are frequently in the milligram and often in the microgram range. Consequently, homogeneity problems with regard to the stable isotopes of carbon and oxygen from cellulose have occurred and these have called for new methods in the preparation of cellulose for reliable isotope analyses. Three different methods were tested for preparing isotopically homogenous cellulose, namely mechanical grinding, freezing by liquid nitrogen with subsequent milling and ultrasonic breaking of cellulose fibres. The best precision of isotope data was achieved by freeze-milling and ultrasonic breaking. However, equipment for freeze-milling is expensive and the procedure is labour-intensive. Mechanical grinding resulted in a rather high loss of material and it is also labour-intensive. The use of ultrasound for breaking cellulose fibres proved to be the best method in terms of rapidity of sample throughput, avoidance of sample loss, precision of isotope results, ease of handling, and cost.

Endocytosed SP-A and surfactant lipids are sorted to different organelles in rat type II pneumocytes.

Intracellular transport of endocytosed surfactant protein A (SP-A) and lipid was investigated in isolated rat type II cells. After internalization, SP-A and lipid are taken up via the coated-pit pathway and reside in a common compartment, positive for the early endosomal marker EEA1 but negative for the lamellar body marker 3C9. SP-A then recycles rapidly to the cell surface via Rab4-associated recycling vesicles. Internalized lipid is transported toward a Rab7-, CD63-, 3C9-positive compartment, i.e., lamellar bodies. Inhibition of calmodulin led to inhibition of uptake and transport out of the EEA1-positive endosome and thus of resecretion of both components. Inhibition of intravesicular acidification (bafilomycin A1) led to decreased uptake of both surfactant components. It inhibited transport out of early endosomes for lipid only, not for SP-A. We conclude that in type II cells, endocytosed SP-A and lipid are transported toward a common early endosomal compartment. Thereafter, both components dissociate. SP-A is rapidly recycled to the cell surface and does not enter classic lamellar bodies. Lipid is transported toward lamellar bodies.

On the applicability of two different stimulation techniques for intra-operative peroneal nerve conduction testing.

Dysfunction of the peroneal nerve is an important complication of knee surgery. We compared two monitoring procedures of peroneal nerve function during a standardized operation, a closing wedge high tibial osteotomy. For two types of stimulation the evoked compound motor unit action potentials (CMAPs) were recorded on the tibialis anterior muscle. We used direct perineural electrical stimulation of the common peroneal nerve distal to the cuff (dCMAPs) after nerve identification in the surgical field. Additionally, magnetic stimulation of the sacral plexus proximal to the cuff (pCMAPs) was performed. It was found that dCMAPs were recorded during almost one hour of tourniquet time whereas the pCMAPs were blocked after 25-30 min in 9 out of 11 cases. On the other hand, the CMAP obtained after proximal stimulation exhibited a latency shift with tourniquet yielding an indicator of ischaemic changes present beneath and distal to the tourniquet cuff. In conclusion, different applicabilities of both stimulation techniques under tourniquet conditions were demonstrated.

Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes.

Surfactant protein (SP) A and SP-A-mediated lipid uptake by isolated type II cells were investigated with biochemical and morphological methods. Inhibition of coated-pit function by potassium depletion severely reduced both SP-A and SP-A-mediated lipid internalization. Lipid uptake in the absence of SP-A was not affected. With confocal laser scanning microscopy and immunoelectron microscopy, SP-A and lipid predominantly (60%) colocalized in intracellular vesicles carrying early endosomal markers (EEA1) 5 min after endocytosis but were negative for the late endosomal or lysosomal marker LAMP-1. As estimated by subcellular fractionation, at this time point, 23% of the internalized SP-A and 45% of internalized lipid were localized within light (<0.38 M sucrose) fractions, which contain lamellar bodies and are positive for EEA1. The remaining label was predominantly found within EEA1-positive and plasma membrane-containing subfractions (> or = 1 M sucrose). We suggest that in isolated type II cells in vitro, SP-A and lipid are taken up together via the coated-pit pathway and that at early time points, both components reside in the same early endosomal compartment.

HDL and vitamin E in plasma and the expression of SR-BI on lung cells during rat perinatal development.

Vitamin E is the most important lipophilic antioxidant, and beneficial effects on oxidant-caused injuries have been reported. Neonates are at high risk of oxidative injury in the lung and other organs because of a low vitamin E concentration, but the optimal timing of the application, a safe application form, and the optimal dosage of vitamin E are not known at present. We recently showed that alveolar type II cells take up vitamin E preferentially from high-density lipoprotein (HDL), probably by means of the candidate HDL receptor, scavenger receptor class B type I (SR-BI; Kolleck et al. Free Rad Biol Med 27; 882-890, 1999). Therefore, both the HDL-bound vitamin E in plasma and the expression of SR-BI on alveolar type II cells may determine the supply of the cells with vitamin E. We show here that the plasma level of vitamin E, total and HDL cholesterol, and the ratio of vitamin E to polyunsaturated fatty acids and to total fatty acids decrease during fetal rat development, reaching the minimum at the postconceptual day 21 (day -1). These parameters increase thereafter to about the same levels as in adult rats. SR-BI is not detectable until day -1 on fetal lung cells, but the expression during the postnatal phase follows the same pattern as the plasma lipid constituents. We conclude that the ability of alveolar type II cells to take up vitamin E develops perinatally in mature neonates. This aspect also has to be considered when the optimal timing of supplementation for the protection of preterm neonates with vitamin E against oxidative lung injury is established.

Surfactant protein A binding to cytomegalovirus proteins enhances virus entry into rat lung cells.

The role of surfactant protein (SP)-A in cytomegalovirus (CMV) infection of the lung was investigated. We found that SP-A binds to various immobilized human CMV proteins and those exposed on the surface of infected embryonal lung fibroblasts. The interaction between SP-A and immobilized CMV proteins was found to be calcium-dependent and inhibited by mannan, suggesting involvement of the carbohydrate recognition domain of SP-A and high-mannose carbohydrate residues of viral envelope glycoproteins. Using flow cytometry and confocal laser fluorescence microscopy in the rat model we showed that preincubation of rat CMV with SP-A stimulates its binding and internalization by rat type II pneumocytes and alveolar tissue macrophages. This effect was concentration- and Ca(2+)-dependent but was not inhibited by mannan. Therefore, the domains of SP-A involved in SP-A CMV interaction and in interaction of the SP-A/virus complex with rat lung cells are distinct. Additionally, in the human CMV model, sheep as well as human proteinosis SP-A did not significantly affect human CMV replication in embryonal lung fibroblasts. Thus, SP-A may contribute to CMV-associated pathology of the lung by increasing the efficiency of target cell infection.

Internalized SP-A and lipid are differentially resecreted by type II pneumocytes.

Biochemical and morphological assays were developed to study surfactant protein A (SP-A) and lipid resecretion kinetics by isolated type II cells in vitro. After a 10-min uptake period with SP-A (3 microg/10(6) cells) in combination with liposomes (60 microg/10(6) cells), the cells were allowed to resecrete. After 5 min of resecretion, only 21.7 +/- 4.6% of the internalized SP-A remained intracellularly compared with 54 +/- 2.9% of the lipids. Extracellular SP-A present during the resecretion period partially inhibited resecretion (SP-A, 36% at 5 min; lipid, approximately 16% at 5 min). Lipid resecretion was also dependent on the SP-A concentration present during the uptake period. Although, as shown by confocal laser scanning microscopy, after a 10-min uptake period at 37 degrees C, most of the fluorescein isothiocyanate-labeled SP-A and rhodamine-phosphatidylethanolamine-labeled lipids colocalized within the cells, after an additional 10 min of resecretion, both the strength of the fluorescence signals and the extent of colocalization had markedly decreased. These data indicate that internalized lipid and SP-A can be resecreted rapidly by type II cells, likely via different pathways.

Inhalation study on exposure to bitumen fumes. Part 1: Development and validation of the equipment.

Bitumen fumes emitted during road paving or roofing contain polycyclic aromatic hydrocarbons (PAHs). Experimental studies have been previously performed to test the carcinogenic potency of bitumen fumes. Some of them have been criticised either on the grounds that the fume condensates were not representative of fumes to which humans are exposed or because the fumes were never characterised in terms of particle size and poorly in terms of composition and concentration in the chambers. For a nose-only inhalation study, we have evaluated the ability of a new fume generation system to deliver stable and reproducible atmospheres of bitumen fumes to an inhalation chamber and investigated the representativity of the fumes generated at a concentration level of 5 mg/m3. The fume generator comprises: (1) an insulated 20 l heated kettle (200 degrees C for bitumen); (2) an insulated inlet pipe with a needle valve to adjust the flow of the test compound from the kettle; (3) a fume generation chamber equipped with a series of interchangeable channels of different width. The fume concentration in the exposure chamber can be controlled by changing the channel width or by restricting the evaporation surface with aluminium foil, and/or by changing the flow rate. Samples of the atmosphere in the chamber were collected and analysed for quantitative determination of total particulate matter (TPM), soluble matter, benzo[a]pyrene (B[a]P) content of the fumes and other PAHs, and evaluation of the particle size distribution. The representativeness of the fumes has been tested by comparison with fumes generated in the Shell small-scale fume rig, which was previously validated against field fumes collected during paving operations. Evaporative losses from the filters during sampling, transport and storage have been also assessed. At 5 mg/m3 TPM, the agreement between laboratories was quite good for the TPM analyses and was good for the soluble matter and B[a]P. Evaporative losses may lead to underestimation of the true exposure level in the inhalation chambers but the use of an XAD-2 cartridge backup is one approach to partially recover losses which occur on the filter. The particle size distributions are somewhat different from those reported for fumes associated with roofing and indoor mastic laying works, in that we found more than 85% of particles to be smaller than 1 micron, compared with 40% particles in the previous analyses. In conclusion, this equipment allows reproducible generation of fumes at the 5 mg/m3 TPM that are fairly representative of those produced in the field with the same bitumen.

Inhalation study on exposure to bitumen fumes. Part 2: Analytical results at two exposure levels.

During the hot application of bitumen-containing materials, e.g. in road paving or roofing, fumes are emitted that contain traces of polycyclic aromatic compounds (PACs). Although worker's exposure to these fumes is low, it might lead to health problems. For studying DNA adduct formation as a consequence of inhalation of bitumen fumes we developed and validated an inhalation system (a dynamic fume generator plus a nose only inhalation chamber). This paper presents and discusses the analytical results from the different laboratories involved in this study on the fumes sampled in the inhalation chamber during three series of experiments where the animals were exposed to fumes at the 5 mg/m3 and 50 mg/m3 level, coming from bitumen heated at 200 degrees C and, as a positive control, fumes from coal tar, heated to 110 degrees C at the 5 mg/m3 level. The following parameters were controlled: temperatures at different key places in the generator; humidity of the chamber; the bitumen or coal tar flow rate; and Total Particulate Matter (TPM). Analyses were performed for Benzene Soluble Matter (BSM), the EPA polycyclic aromatic hydrocarbon (PAH) mixture and for a number of heteroatom-containing PACs. The data show that the coal tar fumes produced at 110 degrees C were very volatile and that most of the differences in particulate matter found between the laboratories can be attributed to evaporative losses. The bitumen fumes boil 25-50 degrees C higher and contain higher boiling compounds. A comparison is made between the PAC exposure profiles for bitumen experiments aimed at 5 and 50 mg/m3. Although the same molecules are found in both fumes their proportion is dramatically different. This effect is largest with the 2- and 3-ring PACs, the ratio of the concentrations found in the 50 mg/m3 TPM concentration to that in the 5 mg/m3 experiment gradually declines from 5500 for acenaphthene to 500 for pyrene, for the 5-ring PACs this ratio is 20-30. As function of their vapour pressure, the ratios of the concentrations of the hetero PACs follow the same trend as that of the 16 EPA PAHs and are of the same order of magnitude. In conclusion, for the compounds investigated, the equipment delivers a fume atmosphere in a reproducible manner. The 50 mg/m3 bitumen fumes are not representatives of field fumes. The reason for these quantitative differences is unclear and further work would be needed to clarify this. Nevertheless it was felt that these fumes at 50 mg/m3 might be a useful tool for qualitative detection of DNA adducts in an animal exposure study.

HDL is the major source of vitamin E for type II pneumocytes.

The alveolar surfactant is a prime target of reactive oxygen species present in air. Alveolar surfactant is supplemented with vitamin E during its assembly in type II pneumocytes. However, it is unknown which of the lipoproteins supply type II pneumocytes with vitamin E. The measurement of the uptake kinetics indicates that HDL might be the primary source of the vitamin E uptake by type II pneumocytes. Vitamin E depletion of rats caused an increase of vitamin E uptake by isolated type II pneumocytes from HDL but not from LDL or VLDL. We demonstrated that type II pneumocytes express the scavenger receptor class B type 1 (SR-B1), an HDL-specific receptor. Vitamin E depletion caused an increased expression of SR-B1 by a post-transcriptional mechanism. The increased vitamin E uptake from HDL and the increased expression of the SR-B1 were reversed by refeeding the vitamin. We propose that HDL is the primary source of vitamin E for type II pneumocytes. The rate of uptake of vitamin E by this cell type might be regulated by the expression of SR-B1.

[Follow-up control of ultrasonographic neonatal screening of the hip]. / Verlaufskontrollen von Hüftbefunden im sonographischen Neugeborenenscreening.

1656 newborns were examined by ultrasound for early diagnosis of CDH in a screening program. In 2.5% of the cases, dysplasia or dislocation of the hip was diagnosed and required treatment. In all cases the final outcome after adequate treatment showed a hip joint without any abnormalities. 11.3% of type IIa-hips (using Graf's classification) did not reach an alpha-angle of 60 degrees until the 12th week. They were then therefore classified as type IIb. 97% of all hip joints were judged as normal in the course of the 4th month. This study emphasizes the necessity of a general ultrasound screening program. It also demonstrated that early and adequate treatment leads to a normal development of the joint.

[The congenital dislocation of the hip joint in ultrasound examination--frequency, diagnosis and treatment]. / Die angeborene Hüftluxation im sonographischen Verlauf--Häufigkeit, Diagnostik und Behandlungskonzept.

BACKGROUND: A real congenital dislocation of the hip joint is very rare. Because of the severeness of the disease an immediate diagnosis and begin of treatment is required. PATIENTS: Between 1984 and 1995 clinical and sonographic screening examinations at 4177 newborns were performed at our hospital. METHODS: We analyzed our patients retrospectively and found out all cases of congenital dislocation of the hip joint. All these cases were classified and the results of treatment were determined. The clinical success of our strategy of treatment was described. RESULTS: Among the 4177 observed newborns 39 cases of congenital dislocation of the hip joint in 27 children were found. We diagnosed the hip type IIIa, IIIb and IV according to Graf in 28.2 and 9 cases respectively. After 12 month a complete healing rate of 95 percent was exhibited with the functional management strategy. Five percent of the affected joints showed a residual dysplasia. The rate of residual dysplasias was 2.5%. CONCLUSIONS: The good prognosis after our early treatment strategy severe congenital dislocation of the hip joint underlines the use of earliest therapy together with a hip screening program.

[Intraoperative computer-assisted measurement of stimulation threshold of the common peroneal nerve in osteotomy of the head of the tibia]. / Intraoperative computergestützte Reizschwellenmessung (Neuromonitoring) des N. peroneus communis bei Tibiakopfosteotomien.

Using a computer-assisted threshold hunting paradigm the motoric threshold of the common peroneal nerve was monitored in 18 patients during a high tibial osteotomy (HTO). The exposed nerve (lateral approach) was stimulated proximal to the osteotomy area and the surface EMG of the M. tibialis anterior was used to guide a threshold hunting device. Motoric threshold as a sensitive indicator of nerve function was found to be almost unaffected by several surgical steps of HTO. Only forceful rotation of a subperiostal Hohmann device during high peroneal osteotomy evoked a slight threshold shift that was fully reversible with device repositioning. The tourniquet, however, affected the threshold significantly. In 10 of the 18 patients the nerve became completely inexcitable after an average time of 59 min. The inexcitability was reversible after opening of the tourniquet. On the other hand, the eight patients maintaining an excitability throughout the entire ischemic period had tourniquet times that did not exceed 60 min. There are several factors that may be responsible for the observed inexcitability after long ischemic periods and we conclude that tourniquet time minimization appears appropriate to avoid neurological deficits during a high tibial osteotomy.

[Intraoperative monitoring of the function of the peroneal nerve in knee joint operations]. / Intraoperatives Monitoring der Funktion des Nervus peroneus bei Kniegelenkoperationen.

Operations on the knee are known to be associated with postoperative neurological complications. There is no consensus opinion on the causes of these complications. The aim of the present study was to develop a method for the intraoperative monitoring of the function of the common peroneal nerve. This was done as to identify intraoperative factors that might be responsible for reversible and irreversible neurological deficits. Computer-aided neuromonitoring is based on online digitizing of the surface EMG of the anterior tibial muscle. An algorithm continuously modifies the amplitude to determine the motor threshold. The method described has been used in 18 patients undergoing high tibial osteotomy. In 10 of the 18 patients, the nerve is rendered completely non-excitable after an average tourniquet application time of 59 min. This non-excitability was reversed on release of the tourniquet. In the remaining 8 patients, excitability was maintained throughout the ischaemic period, which did not exceed 60 minutes in any of the cases. Our method enables accurate quantification of the neural function throughout the entire operation, and convincingly documents the influence of ischaemia on peripheral nerve block.

[Changes in proprioceptive capacities of the shoulder joint in ventral shoulder instability. A comparative study before and after arthroscopic labrum refixation]. / Veränderungen propriozeptiver Fähigkeiten am Schultergelenk bei ventraler Schulterinstabilität. Eine vergleichende Untersuchung vor und nach arthroskopischer Labrumrefixation.

Measurements of movement sense determined by threshold levels for the perception of motion of the shoulder were performed in 10 patients with unilateral posttraumatic shoulder instabilities, in 30 patients who underwent arthroscopic labrum repair for recurrent anterior shoulder instability, and in 15 age-related controls. In patients with anterior shoulder instabilities threshold levels for the perception of motion were higher. In addition, movement sense measurements revealed a marked, but time dependent improve of proprioceptive abilities in patients who underwent arthroscopic labrum repair. While in the shoulders of patients with an postoperative period shorter than 18 months elevated threshold levels for the perception of motion still could be observed, after a postoperative period of 18 moths or more movement sense was not different from normal, uninjured shoulders. In conclusion, our study gives evidence for a proprioceptive deficit with anterior shoulder instability which improves significantly after arthroscopic labrum repair. The restitution of proprioceptive abilities, however, seems to be a time dependent process.

[Gray scale histographic analysis of the acetabular cartilage for quantifying hip ultrasound images]. / Histographische Grauwertanalysen des Pfannenknorpels zur Quantifizierung von Hüftsonogrammen.

AIM: Congenital dislocated hips are classified in ultrasound studies using the angle measuring system of Graf and evaluating the typical changes in acetabular form and structure. We attempted to quantify physiological and pathological changes in the acetabular cartilage using B-mode scan ultrasound studies. METHODS: Gray-level histograms were performed on 894 infant hips; 833 were classified by the Graf scheme as Type IIa and 61 as Type IIIa/b. We compared the histological structure of the cartilage of both the acetabulum and the head of the femur using objective parameters. RESULTS: We could show that gray-level histograms of the acetabular cartilage are an ideal way to study objectively ultrasound evaluations of the same area. Type IIa hips with histographically identified ossification zones show significantly higher healing rates than those without such areas. Type IIIb hips with histographically demonstrated pathological changes showed a significantly longer healing rate and a higher incidence of persistent dislocations that did Type IIa hips with normal cartilage. CONCLUSION: Structural changes in the acetabular cartilage can be evaluated not only qualitatively but also quantitatively to provide prognostic information in infants with congenially dislocated hips. Gray-level histograms nicely supplement ultrasound examination.

[Correlation of pathological clinical hip symptoms in the sonographic study of infant hips]. / Korrelation von pathologisch klinischen Hüftbefunden zur sonographischen Untersuchung von Säuglingshüften.

The relevance of clinical signs of infant hip dysplasia and the importance of a sonographic newborn-hip screening are often discussed. Between 1984 and 1990 at our department 6532 infant hips were examined both clinically and sonographically. Sonographic hip findings were pathological in 71.7% of the hips with positive clinical Roser-Ortolani sign and in 100% of the hips with positive Hilgenreiner sign. In contrast, pathological hip types according to Graf were seen only in 18% of the hips with limitations of the abductions of the hip joint. Examining other clinical signs for infant hip dysplasia within this study, as well we can conclude, that only the Roser-Ortolani sign and that of Hilgenreiner showed a high specificity for infant hip dysplasia, while most of the clinical signs e.g. limitation of abduction in the hip joint are rather unspecific.

[Ultrasound follow-up after surgically managed Achilles tendon ruptures]. / Sonographische Nachuntersuchung nach operativ versorgten Achillessehnenrupturen.

PURPOSE: To evaluate surgically repaired ruptures of the Achilles tendon by means of ultrasonography, employing the classification scheme of Thermann et al. METHOD: 54 patients were examined by ultrasonography after an average interval of 4.4 years. A 7.5 MHz linear transducer was used with a gel approach piece to evaluate the longitudinal and transverse images of the tendon. RESULTS: More than 90% of patients AHD Grade 2 or 3 tendons; in only 3 patients could the repaired tendon be designated Grade 1 or comparable to the healthy control. CONCLUSION: There was no correlation between the relatively unsatisfactory results seen on ultrasonography and the clinical results. We cannot recommend ultrasonography as the only method for assessing postoperative results following Achilles tendon repairs.

SP-A-binding protein BP55 is involved in surfactant endocytosis by type II pneumocytes.

The mechanism of surfactant protein (SP)-A-mediated lipid uptake by rat type II pneumocytes was investigated. In the absence of SP-A, freshly isolated type II pneumocytes actively take up very little if any liposomes. Most of the increase with time is independent of energy or temperature but is most likely due to spontaneous exchange of labeled lipids between liposomes and cell membranes. With 5 micrograms/ml SP-A, type II cells actively take up liposomes (244 pmol dipalmitoylphosphatidylcholine.h-1.10(6) cells-1). The effect of SP-A on uptake is temperature dependent and can be abolished by ATP depletion of the cells. Coincubation with an auto-anti-idiotypic antibody against the SP-A-binding protein BP55 on the cell membrane of type II pneumocytes inhibits SP-A-mediated lipid uptake by type II cells. With increasing amounts of extracellular SP-A present, increasing amounts of liposomes are taken up and directed toward a nondegrading compartment. We suggest that SP-A-mediated surfactant lipid uptake is a receptor-mediated endocytotic process involving BP55.

Identification of a new surfactant protein A binding protein at the cell membrane of rat type II pneumocytes.

Antibodies against a type II pneumocyte component were developed by an auto-anti-idiotypic approach using surfactant-associated protein A (SP-A) as the immunogen. The antibodies recognize an SP-A-binding protein (approximately 170-200 kDa under non-reducing conditions, 55 kDa under reducing conditions) on the cell membrane of rat type II pneumocytes. One of the antibodies competes with SP-A for binding to type II cells. In immunization assays in vitro, with this antibody as the antigen, anti-SP-A antibodies have been generated. The SP-A-binding cell membrane protein recognized by this auto-anti-idiotypic antibody may be involved in the interaction of extracellular SP-A with the type II pneumocyte and may play a role in the regulation of alveolar surfactant metabolism.