Four pedigrees of the cation-leaky hereditary stomatocytosis class presenting with pseudohyperkalaemia. Novel profile of temperature dependence of Na+-K+ leak in a xerocytic form

We report four pedigrees of the group of Na(+)-K(+)-leaky red cell disorders of the 'hereditary stomatocytosis' class. Each showed pseudohyperkalaemia because of temperature-dependent loss of K(+) from red cells on storage of whole blood at room temperature. All pedigrees showed an abnormality in the temperature dependence of the 'passive leak' of the membrane to K(+). Two pedigrees, both of which showed a compensated haemolytic state with dehydrated red cells and target cells on the blood film, showed a novel pattern, in which the profile was flat between 37 degrees C and about 32 degrees C then dropped as the temperature was reduced to zero. The third showed the 'shallow slope' profile, with stomatocytes on the blood film and very markedly abnormal intracellular Na(+) and K(+) levels. Minimal haemolysis was present. The fourth pedigree, of Asian origin, showed the shoulder pattern (minimum at 32 degrees C, maximum at 12 degrees C) with essentially normal haematology. Both of these latter two forms have previously been seen in other pedigrees. The first variant represents a novel kind of temperature dependence of the passive leak found in these pedigrees presenting with pseudohyperkalaemia.     

Two further British families with the 'cryohydrocytosis' form of hereditary stomatocytosis

We describe two families with the 'cryohydrocytosis' form of stomatocytosis. Both show a mild stomatocytic anaemia with Hb levels of 12-16 g/dl and reticulocyte counts of 4.3-24%, with very marked autohaemolysis at refrigerator temperatures and pseudohyperkalaemia as a result of loss of K from red cells on storage at room temperature. The ouabain + bumetanide-insensitive 'passive leak' K influx showed a 'U'-shaped temperature dependence, with a minimum at 23 degrees C. In one family, there was consistent variation in haematological severity within the pedigree. In the other, the parents of the proposita were normal, but all three of her children were affected, consistent with a new mutation of a dominant condition. Cold storage of the red cells led to a very marked increase in osmotic fragility and macrospherocytosis, explaining why a diagnosis of 'hereditary spherocytosis' can easily be reached in these pedigrees.     

Familial pseudohyperkalaemia Chiswick: a novel congenital thermotropic variant of K and Na transport across the human red cell membrane

Two families with inherited abnormalities in Na and K transport across the red cell membrane are described. Both presented with 'pseudohyperkalaemia' as a result of loss of K from the red cells on storage at room temperature. Routine haematology was essentially normal, except for macrocytosis in one family. Studies of the temperature dependence of the passive leak to K showed a novel shoulder pattern with a minimum at 25 degrees C, a maximum at 10 degrees C, followed by a further fall. As in other cases of red cell-based pseudohyperkalaemia, the abnormal temperature dependence of this 'leak' flux could be held to account for the loss of K from the cells at room temperature. These cases represent a novel variant of the temperature dependence of the passive leak of K and Na across the red cell membrane, and can be classified as a mild, non-haemolytic form of the group known as the hereditary stomatocytosis and allied disorders'.     

Successful banking of pancreatic endocrine cells for transplantation

To determine optimal freezing and thawing conditions for rat pancreatic endocrine cells (PEC) and insulinoma cells, five different cryopreservation protocols were compared in this study. PEC and insulinoma cells were cooled at rates of between -0.3 degrees C/min and -5 degrees C/min to -70 degrees C in the presence of 10%, 15%, or 20% dimethylsulfoxide (DMSO) with a programmable temperature controller and then transferred to liquid nitrogen for storage. Frozen cells were thawed by either rapid (in 37 degrees C water bath) or slow (in air) thawing procedure. One hour after the thawing process, cellular viability was determined by trypan blue dye exclusion. The viability results for PEC and insulinoma cells were similar and showed that a slow cooling rate at -0.3 degrees C/min in combination with a rapid thawing in 37 degrees C water bath gave the best results, with up to 80% cellular viability. Cryoprotectant DMSO used at 10% concentration was the most effective among the three concentrations tested. Later, transplantation studies were performed with PEC cryopreserved with the best protocol, which is -5 degrees C/min to 4 degrees C, held for 3 minutes, -0.3 degrees C/min to -7 degrees C, held for 3 minutes, -0.3 degrees C/min to -40 degrees C, and -5 degrees C/min from -40 degrees C to -70 degrees C in 10% DMSO with a programmable temperature controller then transferred to liquid nitrogen for storage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Familial pseudohyperkalaemia Cardiff: a mild version of cryohydrocytosis

We have investigated a Welsh pedigree showing the 'familial pseudohyperkalaemia' phenotype of dominantly inherited, red-cell-based, temperature-dependent pseudohyperkalaemia associated with normal haematology. The 'passive leak' to K across the membrane of these abnormal red cells showed a 'U-shaped' temperature dependence, with a minimum at about 23 degrees C, qualitatively similar to that seen in the frankly haemolytic 'cryohydrocytosis' variant of the hereditary stomatocytosis group. Like three previous pedigrees with cryohydrocytosis, these patients show an excess of ether lipids in the membrane. However, these patients differ from other 'familial pseudohyperkalaemia' pedigrees, in which the leak showed different temperature profiles.     

Complement-Independent Lysis of Human Red Blood Cells by Cold Hemagglutinins

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.     

Acid-base state and intermittent breathing in the torpid bat, Eptesicus fuscus.

The effects of intermittent breathing on acid-base state and blood gases were characterized in the torpid bat, Eptesicus fuscus, during steady-state torpor between body temperatures (Tb) of 5 and 37 degrees C. Arterial blood samples were taken from indwelling catheters without disturbing the torpid state. Arterial pH (pHa) of samples taken without knowledge of ventilatory state rose by 0.15 units from 37 to 5 degrees C with a delta pHa/delta Tb slope over this range of -0.0055 U/degrees C. However, at and below Tb = 20 degrees C, Eptesicus fuscus breathes intermittently with typical apneic periods of 40-150 min and 4-12 min at 10 and 20 degrees C, respectively. Samples taken at the end of a ventilatory bout and near the end of an apneic period at Tb = 20 degrees C revealed cyclic changes in pH (from 7.49 +/- 0.02 to 7.34 +/- 0.01), PO2 (from 96.6 +/- 3.4 to 30.8 +/- 3.9 Torr), and PCO2 (28.2 +/- 1.4 to 45.9 +/- 1.5 Torr). Between 10 and 37 degrees C, end-ventilatory pHa varied inversely with temperature with a delta pHa/delta T slope of -0.011 U/degrees C. Because intermittent breathing is common to many animals during hibernation, these results demonstrate the importance of coordinating blood sampling with ventilatory state for a reliable interpretation of acid-base regulation under these conditions.     

Lymphocytotoxic antibodies in systemic lupus erythematosus: studies of their temperature dependence, binding characteristics, and specificity in vitro.

The lymphocytotoxicity of 33 lupus sera was tested against purified helper/inducer (OKT4) and cytotoxic/suppressor (OKT8) subsets of T lymphocytes at 15 degrees C and 37 degrees C in vitro. There was significantly less killing of both OKT4 and OKT8 cells at 37 degrees C (p less than 0.001 and p less than 0.01) and the ratio of OKT4/OKT8 cell killing at 15 degrees C (1.39 (0.73); mean (SD] was different from that observed at 37 degrees C (0.79 (0.42)) (p less than 0.001). OKT4 killing was greater than OKT8 killing in 21 out of 33 sera at 15 degrees C, while 22 of these sera showed predominantly OKT8 cytotoxicity at 37 degrees C. The relation between the OKT4/OKT8 cell ratio and OKT4/OKT8 serum killing was examined in 22 patients at both temperatures: a significant inverse correlation was observed at 37 degrees C (r = -0.53; p = 0.015) but not at 15 degrees C (p greater than 0.05). The addition of metabolic and cytoskeletal inhibitors increased cytotoxicity at 37 degrees C, but not IgM surface binding. A Scatchard binding analysis of the reaction at 15 degrees C showed that large numbers of antibody molecules were bound to both subsets, with a low average dissociation constant of less than or equal to 6 x 10(-8) mol/l, and electrophoretic blotting indicated that the target surface antigens varied in type and number among individual lymphocytotoxic sera. The demonstration of temperature dependent, tight binding between lymphocytotoxic antibody and variable antigens on the T cell surface emphasises the potential for this phenomenon to affect lymphocyte function in vivo in patients with systemic lupus erythematosus.     

Permeability of membrane to potassium in hypochromic red cells with different specific density.

In hypochromic anaemias (heterozygous beta-thalassaemia and iron deficiency anaemia) ligher red cells lose more K+ than heavier ones, following incubation at 37 degrees C for 24 h. Both in the light and heavy fractions two subpopulations of cells with different permeability to K+ can be separated by a new centrifugation after incubation. On the basis of the results, a relationship between K+ permeability and probability of survival in hypochromic cells is suggested.     

Prostaglandins and CCl4-induced liver cell mortality. The effect of the prostaglandin analogue enprostil

Prostaglandins have been reported to reduce the carbon tetrachloride (CCl4)-induced liver cell injury in rats. The object of the present experiments was to examine the effect of the prostaglandin E2 analogue enprostil on the survival of isolated liver cells exposed to CCl4. Liver parenchymal cells were isolated from rat livers by collagenase perfusion and released into a 'suspension' buffer. Aliquots of the cell suspension were incubated with 1 micrograms or 0.5 microgram CCl4, and to parallel test suspensions 20 ng enprostil was added 5-10 min before CCl4. Incubation was performed on ice, at room temperature, and at 37 degrees C. The average percentage of dead cells after CCl4 treatment was significantly reduced by pretreatment with enprostil at room temperature (1 microgram CCl4: 69 +/- 21% and 44 +/- 13%, respectively) and after 10 min of incubation at 37 degrees C (1 microgram Cl4: 56 +/- 25% and 37 +/- 27%; 0.5 microgram CCl4: 51 +/- 33% and 29 +/- 18%, respectively). When the liver cell mortality approximated 100% after long-term incubation at 37 degrees C, no protective effect of enprostil was observed.     

Cryopreservation of Trichinella spiralis muscle stage larvae

A cryopreservation protocol for Trichinella spiralis muscle stage larvae is described. Larvae are pretreated in 10% bile at 37 degrees C for 1 hr to induce an increase in surface permeability, than incubated in 20% v/v ethanediol at 37 degrees C for 10 min, transferred to 0 degrees C for a second incubation step of 15 min in a v/v mixture of 33% ethanediol: 33% methanol: 34% saline at 0 degrees C followed by rapid cooling (approximately 5,100 degrees C min-1) of aliquots distributed onto glass coverslips. The larvae are thawed by dropping the coverslips into 2 ml saline at room temperature (20 degrees C) and immediately agitating, which simultaneously dilutes (1:100) the cryoprotectants. Groups of 6 mice were infected with muscle stage larvae by gastric intubation. Five days post-infection 14.5 +/- 3.2% of control untreated unfrozen MSL and 16.5 +/- 4.1% of unfrozen bile and cryoprotectant treated controls were recovered as adult worms from the small intestine. Of the cryopreserved larvae, 1.1 +/- 0.4% were recovered as adults, which represents 7.6% compared to the untreated unfrozen controls. When the bile pretreatment step was omitted fewer adult worms (0.09 +/- 0.04%) were recovered and no second generation muscle stage larvae were produced. Modifying this technique by omitting the first incubation step in 20% ethanediol and extending the second incubation step to 25 min yielded 72.3% recovery of T. nativa adult worms 5 days post-infection compared to unfrozen controls. The reproductive capacity index of bile treated cryopreserved T. spiralis was 2.5 +/- 0.6 compared to 51.8 +/- 18.8 for normal muscle stage controls.     

Role of red blood cells in the coronary microcirculation during cold blood cardioplegia

The role of red blood cells during cold blood cardioplegia was studied using an intravital microscope in 13 isolated canine hearts perfused with diluted blood containing potassium chloride. The coronary microcirculation on the left ventricular epicardial surface was observed while the perfusate temperature was varied between 37 degrees C and 10 degrees C. Considerable sludging of red blood cells occurred during hypothermia. The percentage of capillaries perfused by red blood cells (percentage change) significantly decreased as perfusate temperature was reduced (100, 56, and 31% at 37, 20, and 10 degrees C respectively). This was caused by occlusion of microvessels due to sludging and by functional closure due to hypothermia. There was incomplete recovery of perfusion of capillaries at the end of rewarming (60%). The diameters of venules were reduced to 76% of control value at 10 degrees C because of the decrease in the numbers of feeding capillaries, but this value returned to 91% at the end of rewarming. Coronary vascular resistance (mmHg.ml-1.min-1 (kPa.s.litre-1] significantly decreased from 2.0(0.2) at 37 degrees C to 1.2(0.12) at 10 degrees C, but it increased to 2.4(0.24) at the end of rewarming. The finding in this study that sludging occurred which was slow to clear is a definite disadvantage of perfusion with red blood cells during hypothermia.     

Transformation of Chick-Embryo Fibroblasts by Wild-Type and Temperature-Sensitive Rous Sarcoma Virus Alters Adenylate Cyclase Activity

The activity of the enzyme adenylate cyclase, a component of the plasma membrane, has been determined in chick-embryo fibroblasts and in cells transformed by either Bryan high-titer strain of Rous sarcoma virus (RSV-BH) or a temperature-sensitive mutant of this virus (RSV-BH-Ta). Adenylate cyclase activity is reduced in cells transformed by the wild-type virus and also by the temperature-sensitive mutant when the cells are grown at the permissive temperature (37 degrees ). Transformation results in an altered affinity (K(m)) for the substrate (Mg ATP). The apparent K(m) ATP is 0.23 mM in normal cells and 1.1 mM in cells transformed with wild-type virus. The K(m) ATP of the cells infected with RSV-BH-Ta is 0.67-1.0 mM at 37 degrees and 0.28 mM at 42 degrees . The enzyme from normal cells appears to have two binding sites for Mg(++), one at the catalytic site and a second at a regulatory site. Transformation by RSV-BH or RSV-BH-Ta (37 degrees ) apparently alters this second Mg(++) site. A decrease in adenylate cyclase activity occurs within 10 min after cells infected with RSV-BH-Ta are shifted from 42 degrees to 37 degrees ; the activity falls to one-half that of normal cells 30 min after the temperature shift. Our observations indicate that a viral function lowers cyclic AMP content by lowering the activity of adenylate cyclase, probably through some modification of the plasma membrane.     

Comparison of net fluxes of Li and Na in vascular smooth muscle.

The rat tail artery enriched by incubation in a lithium-substituted medium loses Li at three distinct rates upon return to a normal medium at 37 degrees C. The first is very rapid with a single exponential rate constant of the order of 1 min-1, while the second and third are considerably slower with rate constants of the order of 3h-1 and 0.6h-1, respectively. Both slow components are cellular since they combine into a single component when active Na transport is inhibited by the absence of external K, by ouabain, or by low temperature. None of these procedures affects the rapid loss of Li from extracellular locations. It follows that cell Li can be measured simply by washing out extracellular Li at 2 degrees C, a procedure which removes only a small definable amount from cells. The influx of Li into cells during incubation in Li-substituted medium follows 1st order kinetics and exchanges 1:1 with cell K after a delay of about 20 min at 37 degrees C. This delay in influx is associated with active Na transport since it is abolished by the absence of external K, by low temperature, and by ouabain. The activation energy required for the influx of Li and of Na in K-free media is consistent, in the main, the passive diffusion through a restricted area although there is a suggestion in the case of Na that an additional mechanism (e.g. carrier) may be involved.     

Kinetics of Integration of Transforming DNA in Pneumococcus

Integration of donor genes, as measured by recovery of their transforming activity from eclipse in lysates of newly transformed cells of pneumococcus, has been followed at temperatures from 0 to 40 degrees . There is a lag in the recovery curve that is marker-dependent and increases as temperature falls. An Arrhenius plot of the rates shows a sharp break between 15 and 20 degrees . Brief exposure of the system to 37 degrees before incubation at 10 or 15 degrees removes the lag and raises the subsequent rate of recovery. This activation is unstable, however, and disappears when the cells are held at 0 degrees after the exposure at 37 degrees and before incubation at 15 degrees . The results are interpreted in terms of a reaction sequence A right harpoon over left harpoon B --> C, with activation energies for the first forward rate-constant of the order of 50 Cal/mol, for the second, 20-21 Cal/mol, and for the reverse reaction, less than 20 Cal/mol. The properties of the first step, including its marker dependence, are the same as those observed earlier for stabilization of donor markers against intracellular inactivation, and it is suggested they may reflect an activation of the recipient chromosome prerequisite to synapsis.     

Evidence for enhanced sodium transport in the tail artery of the spontaneously hypertensive rat

Transmembrane Na+ and K+ gradients in the rat tail artery were dissipated by overnight incubation in K-free PSS at 10 degrees C and then allowed to recover in normal physiologic salt solution (PSS) at 37 degrees C. The active extrusion of Na+ and uptake of K+ during the recovery period was monitored with Na+ and K+ selective glass electrodes. Passive exchanges were differentiated by re-admitting K+ at 3 degrees C, or in the presence of 1 mM ouabain at both 3 degrees C and 37 degrees C. Active exchange was switched on by an abrupt transfer of the tissue from 3 degrees C to 37 degrees C. Active exchange, measured in perfused, superfused, or sequentially incubated arteries, was distinctly enhanced in young (16-, 20- and 26-week-old) spontaneously hypertensive rats (SHR) of the Okamoto strain compared with age-matched Wistar-Kyoto normotensive (WKY) controls. No such difference was observed in rats with hypertension of 7 or 12 weeks' duration and equal severity induced by unilateral constriction of the renal artery. Steady-state Nai and Ki were measured after washing the tissues for 45 minutes at 3 degrees C in lithium-substituted medium to exchange extracellular sodium with lithium. Cell sodium in these tissues was further partitioned into a free component proportional to [Na]0 and an independent, constrained component. Cell potassium was found to be distinctly elevated in 2- and 4-month-old SHR, while free cell sodium remained normal, despite increased cell permeability demonstrable in a significant exchange of lithium for cell potassium and sodium even at 3 degrees C.     

Effects of pentoxifylline and prostaglandin E1 on cold blood perfusion studied by red blood cell deformability

The effects of pentoxifylline and prostaglandin E1 (PGE1) on red blood cell deformability during cold blood perfusion were studied by means of a filtration method. The deformability of normal red blood cells decreased with the temperature transition from 20 degrees to 15 degrees C (transit time: 3.5, 5.8, 25, and 99 seconds at 37 degrees, 20 degrees, 15 degrees, and 10 degrees C respectively). The effects of both pentoxifylline and PGE1 were not noticeable at 37 degrees C but became significant with hypothermia (5.0, 12, and 48 seconds at 20 degrees, 15 degrees, and 10 degrees C, respectively, in samples with pentoxifylline of 0.3 mg/ml; 5.3, 13, and 46 seconds at 20 degrees, 15 degrees, and 10 degrees C, respectively, in those with PGE1 of 40 ng/ml). The results in this study suggest that pretreatment of blood by pentoxifylline or PGE1 could improve coronary microcirculation perfused with cold blood cardioplegic solutions owing to significant improvement of the deformability of red blood cells.     

Could 37 degrees C storage temperature extend homovital valve allograft viability?

BACKGROUND AND AIM OF THE STUDY: Preservation of allograft valves is the most important determinant of their durability. Unprocessed, homovital valve allografts stored at 4 degrees C in nutrient medium have provided superior mid-term results over routinely used cryopreserved or antibiotic-sterilized allografts. As storage temperature may alter viability, it was hypothesized that allograft storage at 37 degrees C may maintain greater viability over time. METHODS: Porcine aortic (n = 10) and pulmonary valve conduits (n = 10) were harvested under sterile conditions. Valve leaflets and sinus walls were separated, and each was divided into two specimens, which were stored in modified culture medium at 4 degrees C and 37 degrees C, respectively. Cell viability was tested by monitoring metabolic activity at 37 degrees C at days 1, 3, 7, 10, and 14. The proliferative ability of cells isolated from valve leaflets was assessed after 14 days by cell culture. Sterility testing of the storage medium was also carried out. RESULTS: Valve leaflet cells and sinus wall cells had significantly higher metabolic activity when stored at 37 degrees C. The median number of isolated cells at 4 degrees C was 3,231.5 (range: 422-3,844), and at 37 degrees C was 8,317.50 (range: 4,329-8,650). The storage medium was sterile in all cases. CONCLUSION: Storage at 37 degrees C significantly improved valve allograft cell metabolic activity and viability compared with storage at 4 degrees C for up to 14 days. The lower concentration of antibiotics did not affect the sterility of tissues stored at 37 degrees C.     

L428 cells derived from Hodgkin's disease produce E rosette-inhibiting factor

Diminished rosetting capacity of T cells is a well-known phenomenon in Hodgkin's disease, and inhibitors of E rosette formation have been reported to be present in the plasma of patients with Hodgkin's disease. The cell line L428, representing an in vitro counterpart of Hodgkin and Sternberg-Reed cells, could be shown to release a factor capable of suppressing the binding of sheep red blood cells (SRBC) to normal peripheral-blood T lymphocytes or to a T-cell line (L735). At maximally effective concentrations, RIF (rosette inhibiting factor) inhibited T lymphocyte rosetting by approximately 40% (mean from 184 healthy controls). The diminished E rosetting of T lymphocytes from Hodgkin's patients was not further suppressed by added RIF. This factor inhibited binding of SRBC to their target cells at 37 degrees C but not at 4 degrees C. The factor could be stored lyophilized at -20 degrees C and was stable at 56 degrees C (30 minutes). RIF was inactive below pH 6 and above pH 9 or after trypsin digestion. Purification by affinity, ion exchange, and molecular sieve chromatography showed activity peaks at 12.5 Kd, 25 Kd, 50 Kd, and 100 Kd.