Platelet splenic transit times in idiopathic thrombocytopenic purpura. Compartmental vs. non-compartmental model.

Platelet splenic transit times following injection of autologous or homologous 111In-labeled platelets were studied in 42 patients with idiopathic thrombocytopenic purpura. The transit times were determined by two methods from the splenic time-activity curves recorded with a gamma camera: closed two-compartmental model and non-compartmental model (deconvolution analysis). By compartmental analysis the mean splenic transit time for platelets was 6.3 +/- 0.3 min (mean +/- S.E.) and by non-compartmental analysis 7.6 +/- 0.4 min for all cases studied. The mean splenic transit time for autologous platelets was significantly (p less than 0.001) shorter (5.1 +/- 0.3 min) in patients with platelet-associated IgG (measured by platelet suspension immunofluorescence test) than in those with no autoantibodies (7.1 +/- 0.4 min), when the compartmental model was employed. There was no significant difference between mean transit times for autologous platelets in antibody positive and negative patients when deconvolution analysis was applied, but the residue of the splenic transfer function was lower for antibody positive than negative patients (7.2 +/- 1.0% vs. 11.7 +/- 1.6%, p less than 0.05). It is concluded that in idiopathic thrombocytopenic purpura the presence of platelet-associated autoantibodies expands the splenic platelet pool and reduces recirculation of platelets.

Compartmental analysis of short-lived platelet dynamics.

111In-labelled platelets were used for analysing platelet dynamics in 43 patients with idiopathic thrombocytopenic purpura (ITP). The detected time-activity curves, recorded with a gamma camera, were analysed by three methods: two- and three-compartment models, and an open model in which only the splenic curve was analysed. In the two-compartment model the mean rate constant from blood to spleen was 0.328 +/- 0.028 min-1 (mean +/- SEM) and from spleen to blood 0.061 +/- 0.007 min-1, whereas in the three-compartment model the corresponding values were 0.236 +/- 0.020 and 0.044 +/- 0.007 min-1, respectively. The mean rate constant from blood to liver was 0.466 +/- 0.149 min-1 and from liver to blood 0.341 +/- 0.106 min-1 as derived from the three-compartment model. The rate constant from spleen to blood, as determined from the three-compartment model, was significantly higher in patients with a strongly positive result for platelet-associated auto-antibodies (platelet suspension immunofluorescence test (PSIFT] than in patients with a negative PSIFT. The mean hepatic net rate in patients with a high level of antibodies is into the liver, while in patients with little or no antibodies the net rate is into the blood pool. The mean half-life for the fast component of the inverted splenic curve was 2.5 +/- 0.2 min and for the slow component 16 +/- 2 min. In patients with a strongly positive PSIFT the half-life for the slow component was significantly longer than in patients with a negative PSIFT.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 activated lymphocytes (LAK cells) as potential tumor tracers.

The homing characteristics and infiltrative capacity of interleukin-2 activated human peripheral blood lymphocytes, the lymphokine activated killer (LAK) cells, were studied. In vitro stimulated 111In-oxine labeled lymphocytes were injected into the hypogastric artery during hysterectomy, performed because of endometrial carcinoma. Scintigrams demonstrated clear homing of the lymphocytes into the area of the malignant tumor. No selective homing was detectable when labeled red blood cells were injected in a similar fashion. To analyze the infiltrative capacity of the activated lymphocytes, they were incubated in vitro with tumor spheroids grown from cultured glioma cell lines. As revealed by antibodies against the leukocyte common antigen and immunoperoxidase techniques, the activated lymphocytes infiltrated the three-dimensional tumor tissue slowly as a frontier. These results show that, in addition to their previously suggested potential role in cancer therapy, interleukin-2 activated lymphocytes may possibly also be useful as tumor tracers.

Splenic dynamics of indium-111 labeled platelets in idiopathic thrombocytopenic purpura.

Splenic dynamics of 111In-labeled platelets and platelet-associated IgG in 33 patients with idiopathic thrombocytopenic purpura (ITP) were studied. Two half-lives were calculated for the biexponential splenic time-activity curve after i.v. injection of 111In-labeled platelets. There was no difference in the mean half-life of the rapid component of the splenic curve (ST1) when patients with negative or slightly positive platelet suspension immunofluorescence test (PSIFT) were compared to those with strongly positive PSIFT (3.0 +/- 0.7 min vs. 3.6 +/- 0.4, p greater than 0.05). Mean half-life of the slow component of the splenic curve (ST2) was found to be longer in patients with a strongly positive than a negative or weakly positive PSIFT (26 +/- 5 min vs. 13.2 +/- 1.0 min, p less than 0.01). It seems that determination of the two components of the splenic time-activity curve provides a useful method for studying platelet kinetics in ITP.

Sensitivity of 111In-granulocyte scintigraphy in various local infections.

Eighty-eight patients with 105 foci of bacterial infection were investigated by 111In-granulocyte scintigraphy. The overall sensitivity of this method was 74 per cent. A negative correlation between duration of signs and symptoms and the intensity of the granulocyte accumulations was found, and patients having false negative scintigraphy also had a significantly longer history of infection, clearly demonstrating the importance of performing scintigraphy in the acute phase of an infection. Scintigraphy performed after negative or equivocal computed tomography (CT) or ultrasound (US) investigations in patients with strongly suggested occult infection revealed many foci of infection, particularly in areas not covered by CT or US, showing the superiority of 111In-granulocyte scintigraphy over the two other methods when localizing symptoms are absent.

Intestinal accumulation of 111In-granulocytes in patients studied because of occult infection.

111In-granulocyte scintigraphy was performed on 245 patients in whom a localized infection was suspected. In 123 patients scintigraphy was positive and of these 35 (28%) had intestinal accumulations of 111In-granulocytes. Specific local causes for the intestinal uptake of radioactivity were antibiotic associated colitis (eight patients), local pyogenic bowel infection (four patients), systemic disease (two patients), bowel necrosis (two patients), colonic cancer (one patient) and Stevens-Johnson's syndrome (one patient). Nonspecific mechanisms of bowel accumulation were desquamation of labelled granulocytes (12 patients) and bleeding (two patients). In three cases the mechanism of colonic accumulation of granulocytes was not revealed. These results show that unexpected accumulations of labelled granulocytes in the gut is not a rare phenomenon and is often due to clinically significant intestinal inflammation or other disease, especially in patients who do not have signs of respiratory, pancreatic or oesophageal inflammation causing desquamated granulocytes to accumulate in the gut.

Effects of repeated bupivacaine administration on sciatic nerve and surrounding muscle tissue in rats.

The effects of repeated administration of 0.5% bupivacaine or saline into the sciatic notch of rats were studied by light microscopy, electron microscopy and a neurophysiological technique. Very severe myositis, including local necrosis, developed in six of 12 rats treated twice daily with 1 ml bupivacaine for either 3 or 7 days. A 3-h infusion of 1.5 ml 0.5% bupivacaine resulted in minor injury to muscle tissue. A marked degree of disruption and vacuolization of myelin sheaths was evident in nerves exposed to bupivacaine for 3 days. Lymphocyte accumulation was confined to the area surrounding the nervous tissue in 7 of 10 of the preparations from rats treated for 3 days or by a 3-h infusion. No histological changes were detected in nerve and muscle tissue from the opposite extremity exposed to saline. After a recovery period of 3 weeks, no differences in the nerve or muscle histology were seen between samples from bupivacaine- or saline-treated animals. The amplitude of the compound action potential of sciatic nerves was, however, significantly lower after bupivacaine treatment (7 days, 1 ml twice daily). Thus, impaired function may continue despite the lack of histological intraneural injury.

Cytochemical demonstration of estrogen binding sites in breast cancer by estradiol covalently linked to horseradish peroxidase.

17 beta-Estradiol-6-carboxymethyloxime was covalently linked to horseradish peroxidase for the cytochemical demonstration of estrogen binding sites in breast cancer tissue. The affinity of the 17 beta-estradiol-horseradish peroxidase (E2-HRP) conjugate for the estrogen receptor in a human myometrial cytosol preparation was reduced by a factor of about 16 relative to that of 17 beta-estradiol. The estradiol concentration of the E2-HRP conjugate used in the incubations was in the range 2 X 10(-9) -1 X 10(-7) mol/l which, when the reduced affinity of the conjugate is taken into account, corresponds to 1 X 10(-10) -7 X 10(-9) mol/l unbound estradiol. The cytochemical reaction was carried out on cytofuge preparations of cell suspensions of breast cancer tissue. The intensity of the cytochemical reaction was microscopically evaluated by scoring. The results were analyzed in a plot allowing the calculation of an apparent score-max and an apparent Kd value. The reaction intensity was reduced to 20-25% of the control level by a 20-fold excess of 17 beta-estradiol. The cytochemical results correlated positively with the content of estrogen receptors in the cytosol as measured by a validated radioligand method.

Evaluation of the myotoxicity of bupivacaine in bier blocks--a biochemical and electron microscopic study.

The possible myotoxic effect of bupivacaine in combination with tourniquet ischemia was evaluated in 11 patients who underwent surgery of an arm under intravenous regional anesthesia. Eleven patients with the same kind of surgery and tourniquet who had general anesthesia served as controls. Venous blood bupivacaine concentrations in the anesthetized arm were high at the end of tourniquet time (27.2-202 micrograms/m1) and varied from 2.3 to 12.3 micrograms/ml 10 min after tourniquet release. Changes in blood-gas tensions and plasma potassium and lactate concentrations before and just after tourniquet release correlated with the ischemia time. Changes in creatine phosphokinase, lactate dehydrogenase and aspartate aminotransferase activities, possible indices of loss of integrity of muscle cell membranes, varied considerably and did not correlate with the ischemia time. There were no significant differences between the two groups in any of the parameters. Electron microscopy revealed no evidence of muscle degeneration 24 hr after the use of tourniquet with either bupivacaine intravenous regional (n = 4) or general anesthesia (n = 3).

A spin label study on the effect of dibutyryl cyclic AMP on synaptic plasma membranes isolated from rat brain.

The fluidity of the deep layer of the lipid bilayer and the mobility of sulphydryl (-SH) groups of synaptic plasma membranes, isolated from rat brain and spin-labelled with a doxyl derivative of stearic acid and with a nitroxide derivative of maleimide, respectively, were studied using electron spin resonance (ESR) techniques. Dibutyryl cyclic AMP (db-cAMP) and adenosine triphosphate (ATP) had no effect on the fluidity of the deep layer of the lipid bilayer of synaptic plasma membranes. While protein dephosphorylation was inhibited by Zn2+, db-cAMP together with ATP increased the mobility of -SH groups above the value obtained with db-cAMP alone.

Effect of prostaglandin E1, dopamine and dibutyryl cyclic AMP on the fluidity of synaptosome membranes.

The membrane fluidity of synaptosomes, isolated from rat brain and spin-labelled with doxyl derivatives of stearic acid, was studied using electron spin resonance techniques. Prostaglandin E1 fluidized while dopamine ordered both the surface and intermediate lipid layers of the synaptosome membranes. At corresponding concentrations these substances have been shown to modulate synaptic transmission. A possible thermal phase transition of the membrane lipid bilayer was observed at about 35 degrees C (the abrupt change in the Arrhenius plot). Dopamine abolished this thermal change. Dibutyryl cyclic AMP had no significant membrane effects.

Organization of membrane lipids and proteins in human En(a-) erythrocytes that lack the major sialoglycoprotein, glycophorin A. A spin-label study.

Membrane fluidity was studied by electron-spin-resonance techniques in human En(a-) erythrocytes that lack the major membrane sialoglycoprotein, glycophorin A. By using stearic acid spin labels with a doxyl group in the C-12 or C-15 positions, we demonstrated that the hydrophobic core in these cells was more fluid than in normal cells. Surface-located regions in isolated En(a-) membranes, when probed with stearic acid labelled in the C-5 position, appeared more stable than in normal membranes. In isolated En(a-) membranes, protein motion was decreased when probed with a nitroxide derivative of maleimide. After incubation with anti-(glycophorin A) antibodies protein motion and membrane fluidity were increased in normal membranes. This effect was observed also after spectrin depletion, which by itself increased protein motion but decreased membrane fluidity in the hydrophobic core of the membrane. The results show that membrane proteins influence the fluidity of membrane lipids.

Differential nerve block by bupivacaine and 2-chloroprocaine. An experimental study.

The differential blocking effect of bupivacaine and 2-chloroprocaine was studied on isolated rabbit cervical sympathetic trunks (B and C fibres) and phrenic nerves (A fibres). The B fibres (myelinated, preganglionic) were more sensitive than C fibres (unmyelinated, postganglionic) to nerve block by bupivacaine and 2-chloroprocaine, as assessed by changes in action potential amplitude and latency. The blocking action of both bupivacaine and 2-chloroprocaine caused a decrease in amplitude in the B fibres approximately twice as great as that produced in the C fibres. In similar experimental conditions, the fastest conducting A fibres (myelinated, motor) were least affected. Bupivacaine 200 mumol litre-1 completely blocked all B fibres in 7 min and C fibres in 10 min. The average action potential amplitude of A fibres at that time was 78%. 2-Chloroprocaine 300 mumol litre-1 completely blocked B fibres in 4 min and C fibres in 15 min. At 15 min the average action potential amplitude of the A fibres was still approximately 35%. Although these two local anaesthetics differ structurally and physico-chemically, the rates of block of the different fibres were in the same order in vitro.

Classification of acute leukaemia by light and electron microscope cytochemistry.

Blast cells in 33 cases of adult acute leukaemia were investigated for signs of myeloid differentiation by light (Sudan Black B) and electron (peroxidase activity) microscope cytochemistry. All cases which were strongly Sudan positive were also peroxidase positive and electron microscopy is of no further value in these cases. In 11 cases the blast cells were Sudan negative and in 8 cases they were weakly Sudan positive: the results of the electron microscopical analysis would have altered the classification in 9 of these 19 cases. It is concluded that ultrastructural demonstration of peroxidase activity in blast cells is of value in the classification of acute leukaemia in cases with blasts either negative or only weakly positive for Sudan.

The fluidity gradient in erythrocyte membranes in hereditary spherocytosis: a spin label study.

The fluidity gradient and sulphydryl groups in erythrocyte ghost membranes from healthy subjects and from seven patients with hereditary spherocytosis (HS) were studied by spin label techniques. The mobility of the stearic acid spin labels was lower in the spherocyte membranes, indicating greater stability of the phospholipid layer in these membranes than in those of matched controls. This stabilization was most distinct in the intermediate and deep membrane layers. No clear differences were observed in the protein sulphydryl groups. It is suggested that the decreased cellular deformability characterizing spherocytes reflects altered lipid organization or lipid-protein interaction in the interior of the cell membrane.

Membrane characteristics and metabolic properties of glucose-6-phosphate dehydrogenase deficient red cells.

Two Finnish variants of reduced erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity were studied. The G-6-PD Espoo variant is characterized by severe enzyme deficiency which is normally non-haemolytic although primaquine sensitive. The other variant, G-6-PD Helsinki, in which the enzyme activity is moderately reduced, is associated with chronic haemolytic anaemia. The activity of the pentose phosphate pathway was not stimulated by methylene blue in G-6-PD Espoo cells, whereas in normal and G-6-PD Helsinki cells there were increases in shunt activity of 64.5- and 5.3-fold, respectively. As judged by the accumulation of 6-phosphogluconate after incubation with 6-aminonicotinamide, the activity of the pentose phosphate pathway was similar in normal and G-6-PD Helsinki cells, whereas in G-6-PD Espoo cells the metabolic flux through this pathway was decreased. Quantities of sulphydryl groups in intact cells and isolated membranes were similar in normal and G-6-PD deficient cells, as revealed by spin label experiments. In contrast to the situation in normal cells, sulphydryl groups in G-6-PD Espoo cells, and to a lesser extent in G-6-PD Helsinki cells, were sensitive to oxidation by acetylphenylhydrazine. In the G-6-PD Helsinki cells, but not in the G-6-PD Espoo cells, membrane fluidity was increased, as judged from the increased mobility of the stearic acid spin label. Mechanisms are discussed by which G-6-PD deficient cells retain adequate levels of NADPH during resting conditions, and it is suggested that the chronic haemolysis associated with G-6-PD Helsinki could be due to a defect in the lipid region of the cell membrane.

Uptake of radiocalcium by nerve endings isolated from rat brain: pharmacological studies.

1 The uptake of radiocalcium by nerve-ending particles isolated from the striatum of rat brain was studied using lanthanum as a quenching agent. 2 High potassium-induced calcium uptake occurred in two phases: an initial rapid phase and a late slow phase. Following preincubation with CaCl2 2.2 mmol/l for 1 h, dopamine at 1 to 2 x 10(-4) mol/l reduced the high potassium-induced calcium uptake which occurred during the initial rapid phase by 66 and 25% at 2 and 4 s of incubation, respectively, but had no effect on the late slow uptake phase. 3 Haloperidol at 1 x 10(-6) mol/l abolished the inhibitory effect of dopamine on the initial rapid phase of the high potassium-induced calcium uptake. Haloperidol per se had no effect on the calcium uptake. 4 Dibutyryl cyclic adenosine monophosphate at 2.5 x 10(-3) mol/l or prostaglandin E1 (PGE1) at 1 x 10(-5) mol/l had no effect on the initial rapid phase of the high potassium-induced calcium uptake by striatal synaptosomes. Neither of these agents affect calcium uptake by whole brain synaptosomes. 5 It appears that in the striatum, dopamine regulates the depolarization-induced influx of calcium in presynaptic nerve endings. This mechanism could constitute a feed-back inhibition for transmitter release in the striatum.

Uptake of radiocalcium by nerve endings isolated from rat brain: kinetic studies.

1 The uptake of radiocalcium by nerve-ending particles isolated from rat brain was studied in vitro by means of a rapid lanthanum quenching technique. 2 The observed uptake fits a theoretical three-compartment model with two separate uptake phases, a fast, initial phase followed by a late, slow phase. This holds true during control conditions as well as during high-potassium stimulation. 3 The uptake as a function of the external calcium concentration can be described in terms of Michaelis-Menten kinetics during high-potassium stimulation. Under control conditions the fit is clearly applicable but statistically not as good as during potassium stimulation. 4 The affinity for the uptake of calcium remains unchanged under control conditions while during high-potassium stimulation the affinity drastically decreases during the late, slow phase of uptake. 5 During high-potassium stimulation the maximal velocity of calcium uptake is twice that during control conditions. This holds true for both the fast and the slow phases of the uptake. 6 Mg2+ has an inhibitory effect on the uptake, the inhibition being more effective during high-potassium stimulation. Tetrodotoxin has a slight inhibitory effect additional to that extended by Mg2+ during the initial phase of uptake into high potassium stimulated synaptosomes.