A versatile flow cytometry-based assay for the determination of short- and long-term natural killer cell activity.

A flow cytometry based method has been developed to assess natural killer (NK) cell activity in both short-term (4 h) and long-term (18 h) NK assays. Target cells were either labeled with PKH-2, c'FDA or D275. Simultaneously, dead cells were identified by counter-staining with the nuclear dye propidium iodide. Using flow cytometry, only D275 in combination with propidium iodide permits the differentiation of four cell populations: live target cells, dead target cells, live effector cells, and dead effector cells. Even after the extended incubation periods (18 h) necessary for the determination of NK activity in some domestic animals these four populations remain clearly distinguishable. Comparison of results with cells of normal human individuals obtained using this D275/propidium iodide flow cytometry assay with data derived from fluorescence microscopy or an endogenous lactate dehydrogenase release assay shows a strong correlation. Since in long-term NK assays a high proportion of dead effector cells is constantly observed this cell population frequently limits the use of the lactate dehydrogenase release assay but does not interfere with the flow cytometry assay presented here. Using this novel assay, we have demonstrated the suppressive effects of defined glycosaminoglycans on long-term porcine NK activity.

Sulfated polysaccharide anticoagulants suppress natural killer cell activity in vitro.

In contrast to the well studied anticoagulant activity of sulfated polysaccharides (SPS), little is known about their influence on immune competent cells. Using two naturally derived SPSs (unfractionated heparin and low molecular weight heparin), one semi-synthetic SPS (pentosanpolysulfate), and one synthetic SPS (lactobionic acid) as well as the polypeptide hirudin we investigated the effect of these drugs on natural killer cell activity in vitro. We demonstrate that all SPSs tested significantly suppress the activity of natural killer cells at clinically relevant dosages. At all concentrations and at all effector:target ratios tested pentosanpolysulfate was the most potent natural killer cell inhibitor. In contrast, hirudin had no effect on natural killer cell function. Furthermore, scanning electron microscopy revealed that reduced natural killer cell activity is paralleled by decreased lymphocyte cell size and altered cell surface structures. Our results indicate that defined therapeutically applied SPSs can interfere with the tumor cell killing process.

Plasma membrane-integrated estrogen receptors in breast tissue: possible modulator molecular for intracellular hormone level.

Plasma membrane fractions from tumor specimens of 14 peri- and postmenopausal primary breast cancer patients and from non-neoplastic tissue were prepared by sucrose density sedimentation. The membranes were disintegrated by Triton X-100 and the 3H-estrogen binding capacity of membrane-derived proteins was determined. The receptor system found in the plasma membranes was mainly of low affinity and high capacity, working apparently in tandem with the high affinity and low capacity system described for the cytosol. Receptor concentrations in plasma membranes of non-neoplastic and neoplastic tissue were distributed over a wide range of values, but a significantly lowered receptor capacity was found on neoplastic tissue. An association constant, Ka = 6.35 . 10(10) M-1 was determined for neoplasma-derived membrane receptor proteins, whereas non-neoplastic tissue membrane proteins were not saturable, when incubated with up to 150 pmol 3H-estradiol. From a Scatchard plot analysis of some experiments a molar concentration of binding sites for membrane proteins, derived from breast cancer tissue, n(M) = 1.7 . 10(-12)/mg protein was extrapolated. Furthermore, ample evidence was provided by an estrogen fluorescence probe that an estrogen binding system is located within the plasma membrane. It is suggested that the estrogen binding capacity of the epithelium cell membrane, due to the phospholipid moiety, can modulate the estrogen uptake and, thus, preventing hazardous high levels of estrogens within the cytoplasm. The presented experimental data throw a new light on the use of antiestrogens in the treatment of breast cancer.

Monitoring of right ventricular function using a conventional slow response thermistor catheter.

OBJECTIVE: To investigate whether determination of right ventricular end-diastolic volume (RVEDV) and right ventricular ejection fraction (RVEF) can be performed with reasonable accuracy and reproducibility using a conventional slow response thermistor pulmonary artery catheter (CPAC) applying an adaptive algorithm. DESIGN: To study RVEDV and RVEF simultaneously with pulmonary artery catheters equipped with slow and fast response thermistors (FRPAC) under a broad range of cardiac output. SETTING: Laboratory of Institute of Experimental Surgery, Technical University. ANIMALS: 11 anaesthetised piglets. INTERVENTIONS: Hypovolemia (V-) was induced by withdrawal of blood up to 50 ml/kg, hypervolemia (V+) was produced by retransfusing blood and adding up to 30 mg/kg hydroxyethyl starch. In 5 animals in phases V- and V+ beta-adrenergic stimulation was achieved with dobutamine. Finally pulmonary artery hypertension was induced by infusion of small air bubbles. MEASUREMENTS AND RESULTS: Cardiac output (CO), RVEDV and RVEF were determined simultaneously with FRPAC and CPAC placed in the same pulmonary artery branch. Measurements were repeated 8 times sequentially in steady state normovolemia. A total of 130 measurements could be analysed. The coefficient of variation was 6.7 +/- 4.2% for CO(FRPAC) and 4.6 +/- 1.7% for CO(CPAC); for RVEF it was 9.7 +/- 6.2% (FRPAC) and 9.9 +/- 3.9% (CPAC); for RVEDV it was 11.6 +/- 4.8% (FRPAC) and 8.54 +/- 3.2 (CPAC). Mean difference (bias) was 0.06 +/- 0.39 l/min for CO measured with both methods, 19 +/- 35 ml for RVEDV and -3.3 +/- 6.5% for RVEF. CO(CPAC) displayed a strong correlation to CO(FRPAC) (R = 0.97, p = 0.001) as well as RVEF (R for RVEF(CPAC) versus RVEF(FRPAC) = 0.90, p = 0.001). R for RVEDV(CPAC) versus RVEDV(FRPAC) was 0.67, p = 0.001. We conclude that this animal study demonstrates good agreement between RVEF and RVEDV obtained with catheters equipped with a fast response thermistor or with a conventional slow response thermistor allowing accurate monitoring of right ventricular function with a conventional pulmonary artery catheter.

Influence of the beta-1 selective blocker, metoprolol, on the development of pulmonary edema in tocolytic therapy.

Research on one group of nine anesthetized dogs pretreated with alpha-naphthyl-thiourea showed that doses of the beta-sympathicomimetic fenoterol such as those normally administered in clinical tocolysis (2 micrograms/kg per minute) lead to significant fluid displacement to the extravascular space of the lung in the sense of a preclinical interstitial edema. In a second group (N = 9) with the same pretreatment the addition of the beta-1 selective blocker, metoprolol (1.5 micrograms/kg per minute) served to antagonize the hemodynamic changes and possibly the increase of pulmonary capillary permeability induced by the beta-mimetic. Fluid displacement into the interstitium of the lung was prevented by metoprolol. These observations corroborate the hypothesis that pulmonary edema occurring during tocolytic therapy is largely a result of the use of beta-mimetics. In addition to its cardioprotective effect, the administration of the beta-1 selective blocker, metoprolol, may reduce the risk of the development of pulmonary edema in beta-sympathicomimetic therapy for premature labor.

Influence of heparin; of different heparin fractions and of a low molecular weight heparin-like substance on the mechanism of fibrinolysis.

The influence of different heparin fractions and of a synthetic polysulfated polysaccharide (SP54) on the fibrinolytic mechanism was examined. In vitro, a significant shortening of the euglobulin lysis time (ELT) was found after addition of standard mucosa heparin, of high MW heparin and of SP 54 respectively. Low MW heparin fractions had no influence on the ELT. Simultaneously with the shortening of the ELT an activation of factor XII and of kallikrein was observed. A similar effect was found in groups of volunteers after i.v. or s.c. injections and even after oral administration of SP 54. The effect of venous occlusion on the ELT and on the activation of factor XII was considerably increased when heparin or SP 54 was injected 2 h prior to the test. When a comparable concentration of the test substances was added to plasma samples before and after venous occlusion, the effect on the ELT was much less pronounced than after injection whilst the effect on factor XII was comparable in both tests. From these results the conclusion was drawn that activation of fibrinolysis by polysulfated polysaccharides is achieved by an endogenous pathway as well as by an increased availability of the vascular activator. The magnitude of the activation of fibrinolysis partly depends on the MW of the substance but apparently also on the degree of sulfation since a low MW substance with a high number of sulfate bonds such as SP 54 was considerably more active than the low MW fraction of standard heparin.

Fibrinolysis induced by rat glioma cells. A mitosis-triggered process?

Experimentally induced brain glioma cell lines were studied in cell cultures for their fibrinolytic activity; simultaneously the mitotic index was determined. It was found that the most intense fibrinolytic activity of glioma cells coincides with mitotic waves. After 68 hr incubation of SD/G1 cells on a [125I]fibrin layer, [125I]fibrin digestion products were examined by gel chromatography. Hydroxy-urea and actinomycin D were used as inhibitors in cell cultures, investigating their effect on the expression of fibrinolysis. These studies reveal that [125I]fibrin digestion products are mainly low molecular weight (less than 12,000 daltons) compounds and that fibrinolysis is partially inhibited by the drugs added.

Changes in essential fatty acids in plasma lipid fractions of polytraumatized patients with different parenteral nutrition.

We investigated changes in the fatty acid pattern of plasma lipids during the phase of total parenteral nutrition in two groups of polytraumatized patients. For parenteral nutrition we gave L-amino acid solutions in a dose of 0.24 g N day-1kg-1 body weight. In addition, we administered in the first group glucose and fructose, and in the second group glucose, fructose and fat emulsions in a total dose of 30 kcal day-1kg-1 body weight. In the latter group, the proportion of the fat emulsions was 30-40% of the calories administered. The most striking findings were the decrease of the essential fatty acids with regard to the phospholipid fraction from about 60% to 30% in the early post-traumatic phase. In the first group of patients we observed a further reduction of the essential fatty acids in the period of observation. This could be avoided by administering fat emulsions of the same type as we gave in the second group of patients. The functions of essential fatty acids in membranes and in the intermediary metabolism are discussed.

The effect of whole body hyperthermia on 5-fluorouracil pharmacokinetics in vivo and clonogenicity of mammalian colon cancer cells.

In a non-randomized pilot study, 10 patients with histological proof of metastatic colorectal adenocarcinoma were treated by hyperthermia/chemotherapy protocol (41,8 degrees C and 1000 mg 5-FU). Plasma levels of 5-FU were determined after single dose at normothermia and hyperthermia. Plasma concentration time course was consistent with a two-compartment pharmacokinetic open model, with first order kinetic and was significantly altered by hyperthermic treatment. Cytotoxicity of 5-FU was substantially enhanced at hyperthermia (41.8 degrees C) as judged from the clonogenic surviving fractions from established human colon cancer cell lines. Our in vitro and in vivo results suggest that whole body hyperthermia combined with 5-FU is an active combination to develop further in the treatment of colorectal carcinomas.

Changes in essential fatty acids in plasma lipid fractions of traumatized patients.

Changes in the fatty acid pattern of plasma lipid in four different groups of polytraumatized patients were investigated. All of the patients received amino acid solutions containing 0.24 gN/day/kg body weight and 30 kal/day/kg body weight (BW). In group 1, all calories were administered as carbohydrates (glucose and fructose). In group 2, 30 to 40% of the calories were provided as a fat emulsion. When compared to the control group, a reduction in the essential fatty acid concentration in the phospholipid fraction was detected in both groups during the early post-traumatic period. In group 1, a continuous decline was observed during the remainder of the trial period. In group 2, however, the concentration of essential fatty acids remained constant after the initial decline and increased slightly from the 7th day on. In the second part of the investigation, the effect of human growth hormone (HGH) administration on the fatty acid pattern was evaluated. Group 3 and 4 received intravenous feedings identical to the patients in group 2; in group 4, however, 10 mg of HGH per day were added to the infusion. The results of this study confirm the hypothesis that supplemental infusion of a fat emulsion prevents a continuous reduction of essential fatty acids in plasma lipids. An effect of 10 mg/day of HGH on essential fatty acid concentration or composition, however, could not be observed. There were no detectable differences in the percentage of essential fatty acids between those patients receiving and those not receiving HGH.

Proteinases and inhibitors in plasma and peritoneal exudate in acute pancreatitis.

Examination of the peritoneal exudates of 12 patients with acute pancreatitis revealed high activities of pancreatic lipase and amylase. The immunologic levels of the plasma-derived inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in the peritoneal exudates were not markedly different from those of the plasma. However, the inhibitory capacity of alpha 2-macroglobulin, the main inhibitor of human pancreatic trypsin in the exudate, was almost completely depleted when measured by an enzymatic method. Furthermore, spontaneous fibrinolysis occurred in 6 out of 13 exudates applied to plasminogen-free fibrin plates, indicating the presence of free proteinase. This fibrinolytic activity might be inhibited by exogenous alpha 2-macroglobulin or aprotinin (Trasylol, Bayer AG).

Quantification of extracorporeal fibrin deposits in haemodialysis with minimal and conventional heparinization.

The effects of minimal intermittent heparinization and of conventional heparinization on endogen fibrinogen concentration as well as on fibrin deposits were studied on 17 on patients hemodialysis. It was shown that the minimal intermittent heparinization did non reduce dialysis efficiency and that a total heparin dosage of 6,000 units is the lowest safe limit.

Myocardial salvage prior to emergency coronary bypass surgery for PTCA-induced coronary occlusion.

The possibility of prolonged, selective coronary perfusion via angioplasty catheters was evaluated. A pump was developed featuring minimal hemolysis even at high pump pressures. Pressure requirements differed greatly between various catheter types. Catheter-induced hemolysis did not correlate with pump pressure and was mainly due to a turbulent jet effect through sideholes. In 20 of 22 closed-chest dogs, myocardial ischemia could be greatly reduced or completely prevented for over 2 hours after proximal LAD or Cx occlusion. With flow rates between 1 and 2.5 ml/min/kg body weight and using the ECG and coronary venous O2 saturation for rate adjustment, pressure within the selectively perfused vessel never reached critical values. Thus, with the appropriate equipment, prolonged coronary perfusion is feasible. In some cases of percutaneous transluminal coronary angioplasty-(PTCA) induced coronary occlusion, it may prevent ischemic myocardial damage between PTCA-induced acute coronary occlusion and subsequent surgical revascularization.

[X-ray transparency of common orthopedic casts]. / Die Röntgenstrahlentransparenz gebräuchlicher stabiler Stützverbände.

X-ray permeability is one of the important criterions which qualify a material as orthopedic cast. By using a phantom (amputated human lower leg) standardized conditions were created to compare the five customary casts: Cellamin, Lightcase II, Hexcelite, Neofrakt and Baycast. For judging the perceptibility of osseous structures, especially the details of the fracture, the lower and the upper number of clinically used layers were considered. The blank tested valuation showed the well-known advantages of the plastic materials in comparison with plaster of Paris. Within the group of the plastic materials distinct differences were also found.

[Micromorphological and functional alterations of the kidney during persistent ureteral obstruction (author's transl)]. / Mikromorphologische und funktionelle Veränderungen an der Niere bei der chronischen Harnstauung.

Alterations on the upper urinary-tract and on the kidney have been investigated by histology and scanning-electron-microscopy (SEM) in an animal study on the rat. Examinations have been performed 7, 14, 28 and 56 days after double-ligating the ureter. The documentation shows the developed alterations which are remarkable in the area of the papilla and medulla of the kidney, whereas the glomerula are nearly unchanged in comparison with the controls. At the same time function-tests with radioactively labelled Jodium-Hippuran and technetium-DTPA have been performed. The nephrograms in the region of interest technique showed after 14 days isosthenuria and after 4 weeks no signs of any function on the operated side.

[Microsurgical nerve and blood vessel sutures with resorbable suture material]. / Mikrochirurgische Nerven- und Gefässnähte mit resorbierbarem Nahtmaterial.

An experimental study of the behavior of the absorbable suture Polyglactin 910 (Vicryl) 0,2 metric in the tissue was performed. Microsurgical sutures of nerves and vessels (sciatic nerve and carotid artery) were performed and compared with a non-absorbable suture (Ethilon). The micromorphological studies included a period of 12 weeks after surgery. The absorbable suture showed equal mechanical qualities, with the advantage that the suture material and the tissue reaction disappeared after the 10th week, while the poor visualization of the absorbable suture under the operating microscope was a disadvantage.

[Recent aspects of the treatment of local hydrofluoric acid erosion]. / Neue Aspekte in der Behandlung lokaler Flusssäureverätzunger.

In experimental studies we compared the intraarterial injection of calcium to local infiltration of calcium as treatment of local hydrofluoric acid burns. We showed that intraarterial injection of calcium is the superior therapy in terms of earlier and more effective arrest of destruction. The results are better the earlier the injection is given. There were no local or systemic disturbances due to the intraarterial injection of calcium.

[Extreme loading as cause of aseptic loosening of the total hip endoprosthesis socket and the resultant therapeutic consequences]. / Extrembelastungen als Ursachen für aseptische Lockerungen von Hüfttotalendoprothesen--Pfannen und daraus resultierende therapeutische Konsequenzen.

An analysis of the extent of damage in explanted endoprostheses of the hip showed massive destructions of the implanted components, such as fractures of the socket etc. For this reason, the authors designed a mathematical model of the extremity for which a prosthesis is designed; this model can simulate, inter alia, extreme loads of the joint occurring, for example, when a person falls. Self-experiments were helpful in determining the load exercised on the hip joint under such extreme conditions and in defining the effects on implanted joint material and the interface between bone and bone cement. Besides the influence of the body weight and the height of a fall, we also investigated dependence on attenuation promoted by different kinds of shoeware. These extreme loads in everyday life, which result in an excess stress of the materials and the interface, provide a good explanation for the aseptic loosening of the primarily well-fixed socket. From the therapeutic point of view these results suggest that such extreme burdening should be avoided during the time in which repair processes take place to gradually reduce the heat necroses in the osseous bed of the acetabulum which are due to the cement, by exercising a partial load only for three months after the operation rather than applying the full load too early. Attenuation of shock achieved by the use of various shoe materials was studied in self-experiments in which the researcher allowed himself to fall from different heights on to an outstretched leg while wearing different types of shoes.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fulminating malignant hyperthermia in swine--an alternative therapy concept]. / Die fulminante maligne Hyperthermie beim Schwein--Ein alternatives Therapiekonzept.

An inexpensive therapeutic concept compared to the dantrolene-therapy to counter the malignant hyperthermia (MH) is to be discussed, using a case-study from swine-anaesthesia. Hyperventilating the animals (with O2), administering metamizol, beta-blocker, bicarbonate and sufficiently cooling the patient can, if the symptoms are recognized early enough, arrest the hypermetabolic cascade in its track. All the animals that were treated according to this scheme survived the (MH) crisis without any lasting post operative damage.