Differential immunoresponses following experimental traumatic brain injury, bone fracture and "two-hit"-combined neurotrauma.

OBJECTIVE AND DESIGN: Cytokine-mediated immunoresponses are consequences of isolated traumatic brain injury (TBI) and muskuloskeletal trauma but little is known when both impacts occur simulanteously in combined neurotrauma (CNT), i. e. TBI + muskuloskeletal trauma (bone fracture). MATERIALS AND METHODS: A "two-hit"-experimental model of CNT (TBI + tibia fracture) was used to investigate circulating cytokine interleukin-1-beta, -6, -10 and sTNF-R1 concentrations following peripheral bone fracture only, TBI only and CNT. Blood samples were drawn at 30 min, 6 h, 24 h, 48 h, and 7 days following trauma and circulating cytokine concentrations were determined via immunoassay. RESULTS: Circulating cytokines were increased after trauma (p <0.001 vs. controls), but peaked at different time points. sTNF R1 peaked first at 30 min, followed by IL-6 at 6 h after trauma. IL-10 levels were highest at 24 h, and those for IL-1beta at 48 h after trauma. Circulating IL-6 and IL-10 levels were highest in CNT at 8/10 time points studied (p <0.001). CONCLUSION: Circulating cytokine IL-1-beta, -6, -10 and sTNF-R1 concentrations are increased after trauma (TBI, fracture and CNT) but peak at different time points. Pronounced IL-6 and IL-10 responses after CNT may contribute to the increased susceptibility for complications in CNT versus monotrauma.

[Cardiorespiratory effects of heliox using a model of upper airway obstruction]. / Kardiorespiratorische Effekte von Heliox an einem Modell der oberen Atemwegsobstruktion.

Heliox is a mixture of Oxygen and Helium. The low density of Helium allows this mixture to flow in a laminar pattern where oxygen, nitrogen or air flow would be turbulent. Therefore the force necessary to move a volume of gas (e.g. Heliox) is greatly reduced in comparison to a turbulent gas flow. In a respiratory loading experiment we investigated the effects which Heliox exerts on hemodynamic as well respiratory variables. 10 volunteers were breathing spontaneously and through three different endotracheal (ET-) tubes (ID 4.0, 4.5, 5.0 mm). The subjects were switched from room air to Heliox and differences in the variables heart rate (HR), blood pressure (BP), stroke volume (SV), stroke index (SI), peripheral vascular resistance (TPRI) and left ventricular work index (LVWI) were measured. Furthermore the (PhAng) between abdomen and thorax was detected using respiratory inductance plethysmography (n=2) and the sense of dyspnoe under the different conditions was assessed by the use of a dyspnea score (DS). The means of BP, SV, SI, TPRI and LVWI did not significantly differ between the resting and the different loading conditions irrespective of the gas that was used. The variability of hemodynamic measures was significant larger during loaded vs. unloaded breathing. Heliox could significantly reduce this degree of variability. In two subjects Heliox could also significantly reduce the PhAng as well as DS. Heliox showed effects on hemodynamic as well as respiratory and subjective variables. These effects can be interpreted as a reduction of the extent of pressure variations in the intrapleural space leading to less impact on hemodynamic variables while breathing Heliox vs. room air in a resistive loading experiment. In the future the combined measurement of hemodynmic variables as well as non-invasive assessment of respiration might shed new light on cardio-respiratory interaction and effects of Heliox during airway obstruction.

The right ventricle in Fabry disease.

AIM: Left ventricular (LV) hypertrophy is a common feature in Fabry disease-related progressive infiltrative hypertrophic cardiomyopathy and affects both men and women, but at different ages. To date, however, little is known about the role of right ventricular (RV) function in Fabry disease. Therefore, this study aimed to investigate the extent of RV involvement in patients with Fabry disease. METHODS: Echocardiographic examination of the right and left ventricle was carried out in 129 patients (80 women and 49 men) with Fabry disease. RESULTS: RV hypertrophy was present in 46 patients (35.7%). Of these patients, 13 showed signs of severely depressed right systolic function (tricuspid annulus movement < 10 mm and a prolonged RV pre-ejection period/pulmonary ejection time ratio) and six patients showed additional severe depression of parameters of diastolic function (pseudo-normal or restrictive RV filling pattems). Those patients with RV hypertrophy and severely compromised systolic and diastolic function had the highest LV masses (92 +/- 11.7 g/m(2.7)). CONCLUSION: RV involvement is common in Fabry disease and ultimately progresses to severe systolic and diastolic RV dysfunction. These findings might explain why patients with preserved LV function can develop clinical features such as reduced exercise capacity, organomegaly and lymphoedema.

[Duration of induced seizures during selective pharyngeal brain cooling]. / Dauer epileptischer Anfälle während selektiver pharyngealer Hirnkühlung.

Whole body hypothermia can be used to treat the injured brain (e.g. after hypoxic events). Side effects include hemodynamic instability, coagulopathy and infection. Because of these side effects it appears reasonable to cool the brain selectively (selective brain cooling, SBC) without changing the core temperature. A new animal model was used to demonstrate SBC from the pharynx and to examine effects of SBC on the duration of pharmacologically induced seizure activity. Sprague-Dawley rats (n=18, 12 successful experiments) were sedated and mechanically ventilated. Invasive blood pressure monitoring was instituted and blood gases were drawn to evaluate the arterial blood gas status. Electrical brain activity was recorded using a microneedle in the extracellular compartment of the rat brain cortex. Cooled water was circulated through a small tubing into and out of the pharynx of the animals. The cortical as well as the rectal temperature were recorded. After the injection of a single dose of bicuculline (1 mg/kg i.v.) per animal the duration of the induced seizure activity was measured and compared with the temperature prior to the induction of seizure activity. The cortical blood flow (CBF) was detected using intra tissue Doppler signals in the rat cortex in the same location as the EP-study. The influence of a brain temperature reduction between 36.5 degrees to 31.5 degrees C on the seizure duration was examined. There was a positive correlation between the seizure duration and the cortical temperature (r=0.64). Also the CBF was increased during seizure activity (p=0.02) and the increase correlated weakly with cortical temperature (r=0.18). The core temperature remained in the normothermic range (36.9+/-0.7 degrees C) Conclusion: The duration of induced seizures correlates with local brain temperature. In the future further studies should examine the efficiency of induced (selective) brain cooling to treat prolonged seizure activity.

[Selective pharyngeal brain cooling]. / Selektive pharyngeale Hirnkühlung.

Whole-body cooling can be used in the treatment of various brain pathologies, for example, after hypoxic events. Potential complications include haemodynamic instability, coagulation disorders and infection. Selective brain cooling (SBC) would therefore appear to make good sense. In an animal model a new approach to SBC was therefore evaluated. A rat weighing 350 g was sedated with alpha-chloralose (40 mg/kg/h) and d-tubocurarine (4.05 mg/kg/h), mechanically ventilated and placed on a heating pad. A thermocouple was introduced into the somatosensory cortex to a depth of 2.5 mm. SBC was achieved using a novel approach: PTFE tubing (ID 100 microns) with an inlet and an outlet was wrapped around and glued to a piece of wood, and introduced non traumatically into the pharynx. The tubing was perfused with cold water (+4 degrees C). Under SBC the cortical temperature dropped from 38.4 degrees C to 27.7 degrees C while the core temperature remained stable. In an animal model SBC was successfully accomplished via the pharynx. Further studies should now be done to evaluate the effectiveness of this approach in larger animals with potentially different anatomical features.

[Measuring opening pressure in lumbar puncture--a new method]. / Bestimmung des Eröffnungsdruckes während der Lumbalpunktion--ein neues Verfahren.

Determination of the opening pressure (OP) during diagnostic lumbar puncture (LP) yields additional information that may impact on treatment and prognosis in disorders affecting the central nervous system (e.g. meningitis). Established methods contain systematic errors as well as risks to the patient. We therefore present a new procedure that allows measurement of the OP by timing the flow of cerebrospinal fluid through a capillary attached to an LP needle. A resistance located between needle and capillary slows down the flow of cerebrospinal fluid so that it becomes independent of the capillary forces acting on it. The time required for the fluid to travel between two marks on the capillary (defining a given volume) can be used to calculate the flow. Since the combined resistance of needle and resistance can be calibrated, the pressure driving the flow--in this case the opening pressure--can be calculated. A simple model was used to evaluate the impact of different resistances and different needles on OP determination. The effects of cellular elements and proteins in the CSF are discussed.

[Development of a flexible cardiorespiratory monitor based on induction plethysmography]. / Entwicklung eines flexiblen kardio-respiratorischen Monitors auf Basis der Induktionsplethysmographie.

Induction plethysmography (IP) utilizes changes in the inductance of sinusoidal wires embedded in elastic bands placed around the chest and abdomen to detect volume changes in the two compartments. These changes can be attributed to respiration or heart beat. To date, most applications have been tailored to an investigation of respiration. More sensitive systems have been employed for the detection of cardiac activity. The wires within the bands, which function as the coil in a resonant circuit, are excited by an oscillator. Among other factors, the inductance of the coil depends on the cross-sectional area of the coie, and changes with respiration in coils placed around the chest and abdomen. Using LabView software, the biosignals obtained undergo an analog-to-digital conversion prior to processing. The system was calibrated using the isovolume method. In 10 adults, IP was tested against a pneumotachograph (PNT) in different body positions (standing, sitting, supine, prone). Correlation between tidal volumes measured with IP and PNT was of r > or = 0.96 on average, recalibration being done after each change in position. The absolute mean error ranged between 3.7 and 8.5%, depending on body position. The smallest error (3.7%) and greatest agreement between the two methods was found in the supine position (93.3% of the IP measurements within +/- 10% of the PNT measurements). An IP application that could be used to collect data over the long term and which is in good agreement with PNT was developed by employing a "virtual instrument" (VI, LabView) for flexible data acquisition and data processing. Agreement was best when the volunteer adopted a supine position. A smaller correlation was found in standing or seated subjects. This might be due to the fact that in the latter two positions, the respiratory system may have more than 2 degrees of freedom, and thus cannot be adequately monitored by only two bands around the thorax and abdomen. Signals produced by cardiac activity were detectable on the surface of the body.

Mutations in the basic core promotor and the precore region of hepatitis B virus and their selection in children with fulminant and chronic hepatitis B.

The involvement of precore stop codon 1896-A and base exchanges in the AT-rich region at positions 1762 and 1764 of the hepatitis B core promotor has been controversely discussed in adults with fulminant hepatitis B. Because no data are currently available on children, we analyzed the basic core promotor (BCP) and precore region in children with chronic and fulminant hepatitis B. The BCP and precore region were sequenced directly and after cloning from mothers and infants. Thirteen children suffered from chronic liver disease, 6 of whom were treated with interferon alfa (IFN-alpha). All 13 patients seroconverted from hepatitis B e antigen (HBeAg) to hepatitis B e antigen antibodies (anti-HBe), and sera were analyzed before and after seroconversion. Nine vertically infected infants developed a fulminant course of hepatitis B. The occurrence of BCP (1762-T/1764-A, 7.7%) and precore (1896-A, 7.7%; 1899-A, 15%) mutations in chronic hepatitis B was rare. A genotype shift from D to A was observed in 3 patients after development of anti-HBe. A high number of base exchanges was detected in those infants with fulminant hepatitis B. Eight of nine showed a G-A exchange at positions 1896/97 (89%), 1899 (56%), and/or mutations at nucleotide (nt) positions 1762 (56%) and 1764 (78%). All virus strains belonged to genotype D, whereas in the only surviving infant, a D-to-A shift was detected. Hepatitis B virus (HBV) DNA clones were examined from 3 babies and 5 mothers. Our results showed a heterogeneous virus population in 4 of 5 mothers. In contrast, a homogeneous virus population emerged in the infants. According to our data, the analysis in children with fulminant and chronic hepatitis B revealed a striking presence of BCP and precore mutants in infants with fulminant hepatitis (FH) when compared with clinically inapparent anti-HBe-positive children (P <.002), which could be one factor in the pathogenesis of fulminant hepatitis B in children.

[A new microprocedure for continuous and non-consuming determination of cellular oxygen uptake based on fluorescence quenching]. / Ein neues Mikroverfahren zur kontinuierlichen und verbrauchsfreien Bestimmung der zellulären Sauerstoffaufnahme auf Basis der Fluoreszenzlöschung.

The oxygen uptake in a cell suspension can be measured by various methods using manometric, paramagnetic or photometric techniques, oximetry, mass spectrometry or radiospectrometry. Easy-to-apply Clark-type electrochemical (polarographic) sensors are by far the most commonly used devices in medical applications. One of their drawbacks is the fact that they consume oxygen and may cause systematic errors when measuring oxygen uptake. Since the beginning of this century, concentration dependent quenching luminescence by oxygen has been used in a number of experimental settings. Using this analytical approach it is possible to detect oxygen without consuming it. We report about a new method of assessing cellular oxygen uptake using the luminescence quenching by oxygen. In an 850 microliters oxygen-tight microchamber, a fluorescent dye (tetraphenylporphyrin) adsorbed on a monolayer of gas chromatographic beads is separated from a cell suspension by a silicone membrane. An active electrochemical electrode integrated within the chamber is used to calibrate the fluorescence signal. Fluorescence is generated by green light (wavelength lambda = 546 nm), the intensity of the emitted red fluorescent light (lambda > 630 nm) is measured with a photomultiplier tube. As the first application of this new method, the oxygen uptake of human lymphocytes was determined. The cells were prepared using a routine separating technique-gradient centrifugation in Ficoll. For methodological reasons, all experiments were carried out at a temperature of 22 degrees C. In 7 consecutive measurements, an oxygen uptake of 2.81 +/- 0.85 mmol O2/10(11) lymphocytes/h was found. In less concentrated suspensions this figure is higher--an effect known as the "crowding phenomenon"--which means that with increasing cell concentration the specific oxygen uptake rate decreases. Our values for cellular oxygen uptake are higher than those in the literature. Since most reported studies on lymphocytes were done at 37 degrees C, a larger difference between our values and those in the literature would be expected. This may in part be attributed to different cell concentrations, separation techniques, etc. Another explanation might be the fact that electrochemical oxygen sensors used by other authors consume oxygen. Cell suspensions investigated with polarographic sensors therefore need to be stirred. Slow stirring reduces the values of oxygen uptake, so that a high stirring frequency is needed to obtain correct results (so-called stirring effect of polarographic electrodes). High stirring frequencies, however, destroy cells, which might be another factor explaining the results reported above. Our new method based on fluorescence quenching consumes no oxygen, and is therefore independent of stirring.

Mutations in the mu heavy-chain gene in patients with agammaglobulinemia.

BACKGROUND: Most patients with congenital hypogammaglobulinemia and absent B cells are males with X-linked agammaglobulinemia, which is caused by mutations in the gene for Bruton's tyrosine kinase (Btk); however, there are females with a similar disorder who do not have mutations in this gene. We studied two families with autosomal recessive defects in B-cell development and patients with presumed X-linked agammaglobulinemia who did not have mutations in Btk. METHODS: A series of candidate genes that encode proteins involved in B-cell signal-transduction pathways were analyzed by linkage studies and mutation screening. RESULTS: Four different mutations were identified in the mu heavy-chain gene on chromosome 14. In one family, there was a homozygous 75-to-100-kb deletion that included D-region genes, J-region genes, and the mu constant-region gene. In a second family, there was a homozygous base-pair substitution in the alternative splice site of the mu heavy-chain gene. This mutation would inhibit production of the membrane form of the mu chain and produce an amino acid substitution in the secreted form. In addition, a patient previously thought to have X-linked agammaglobulinemia was found to have an amino acid substitution on one chromosome at an invariant cysteine that is required for the intrachain disulfide bond and, on the other chromosome, a large deletion that included the immunoglobulin locus. CONCLUSIONS: Defects in the mu heavy-chain gene are a cause of agammaglobulinemia in humans. This implies that an intact membrane-bound mu chain is essential for B-cell development.

[An innovative procedure of oxygen detection in medicine, biology, environmental research and biotechnology based on luminescence quenching]. / Ein neuartiges Verfahren der Sauerstoffdetektion für Medizin, Biologie, Umweltforschung und Biotechnik auf Basis der Lumineszenzlöschung.

For most (aerobic) animal organisms, oxygen is a mandatory and global substrate. The accurate measurement of oxygen is therefore of importance in the fields of medicine, biology, environmental research and biotechnology. The fact that oxygen is not readily soluble in aqueous media makes its detection more difficult. In contrast to the technique of polarography, the use of luminescence quenching by paramagnetic oxygen, does not consume the oxygen. Another problem of oxygen detection in connection with respiration is the need for very short response times. A third problem, which is associated with luminescence itself, is the fading of the dyes, which results in long-term signal instability. The last two problems can be optimally resolved by adsorbing the luminescence dye onto chromatographic materials--in particular hydrophobic material--having a very large internal surface area, and using the decay time in accordance with the Stern-Volmer equation as oxygen signal. For this, continuous evaluation of the signal is necessary. The carrier material doped with dye is incorporated in a single-grain layer. For measurements in liquids, the detector layer is protected by a black silicone membrane. Two designs are possible for the oxygen detector: (I) a special form using glass fibres, and (II) a miniature detector utilizing optoelectronic solid state technology. Both fluorescence and phosphorescence can be employed, the dye used being excited by light, obviating the need for quartz. The detector layers may be either of high sensitivity for small oxygen concentrations, or have equal sensitivity over the entire oxygen concentration range. There is an optimal figure for the specific amount of adsorbed dye. Application examples are given for respiration and for the determination of oxygen uptake by suspended cells.