Electrophoresis of lymphocytes from normal human subjects and patients with chronic lymphocytic leukaemia

The electrophoretic mobility (EM) of peripheral blood lymphocytes from twelve normal subjects and four patients with chronic lymphocytic leukaemia (CLL) was studied in relation to the thymus-derived (T) lymphocytes and bone marrow-derived (B) lymphocytic system. An attempt was made to correlate electrophoretic results with other methods of identifying T and B lymphocytes—T cells by the formation of sheep-cell rosettes and B cells by the formation of erythrocyte–antibody–complement (EAC') rosettes and by staining for surface immunoglobulin.

In the normal subjects the majority of cells migrated quickly with a small `tail' of slower cells. It is suggested that the faster populations are T cells and the slower, B. In CLL the majority of the cells were slow migrators. There was agreement between the percentage of fast cells as assessed by electrophoresis with that of T cells by sheep-cell rosetting; there was also some agreement between the percentage of electrophoretically slow cells to B cells by EAC' rosettes or surface immunoglobulin.

It was possible to remove some of the B cells by density centrifugation after forming EAC' rosettes. This further defined the T cell peak on electrophoresis.

It is concluded that T and B cells carry different surface charge densities which permit them to be separated by electrophoresis and that the malignant B lymphocytes of CLL migrate electrophoretically in a similar fashion to normal B cells.

     

Diphenylhydantoin and fragility of erythrocytes in normal subjects and in patients with hereditary spherocytic anemia.

To study the effects of diphenylhydantoin (Dilantin, DPH) on membrane function, DPH was incubated with erythrocytes from normal individuals and from patients with congenital spherocytic hemolytic anemia and osmotic fragility measured. The resulting curves show that DPH reduces the osmotic fragility of normal and spherocytic erythrocytes. In the presence of ouabain, DPH still had a protective effect on erythrocytes even though ouabain alone increases hemolysis of erythrocytes at certain saline concentrations. This study shows that erythrocyte fragility can be manipulated in vitro and provides a basis for studies in vivo.     

Transfer factor: failure to transfer reactivity in normal human subjects.

Transfer factor was prepared from highly selected normal donors. One lot was made from donors strongly reactive to coccidioidin and negative to Dharmendra antigen in in vivo and in vitro testing. The other lot was made from donors without reactivity to coccidioidin and strongly reactive to Dharmendra. Aliquots of each lot were injected into eight normal recipients. Eight additional normal recipients were given placebo injections. Before and after injection, skin test reactivity and in vitro testing were evaluated by an individual who did not know which preparation the patient received. Changes in immunologic reactivity in subjects receiving transfer factor could not be distinguished from those in subjects receiving placebo. I conclude that transfer factor does not cause enhancement of immunologic reactivity in normal subjects. A well designed, critical study is needed to determine whether or not it does, in fact, cause enhancement of immunologic reactivity in patients with impaired cellular immune reactivity.     

Elastic properties of peripheral arteries in heart failure patients in comparison with normal subjects

Understanding the change in elastic properties of peripheral arteries in heart failure patients is of particular importance, especially when compared with normal subjects. To investigate factors associated with their difference, 40 normal subjects and 60 heart failure patients were studied. Electrocardiograms, carotid pulses and radial pulses were simultaneously recorded to determine carotid-radial pulse transit time (carotid-radial PTT), arm pulse wave velocity (PWV), and arterial volume distensibility. In comparison with normal subjects, carotid-radial PTT was lower by 8 ms in heart failure patients, arm PWV higher by 1.4 m/s, and peripheral arterial distensibility lower by 0.04 % per mmHg (all significant, P < 0.01). Peripheral arterial distensibility was significantly related to systolic blood pressure (SBP) and to left ventricular ejection fraction (LVEF) for heart failure patients (both P < 0.001), but the relationship for the normal group was not statistically significant (both 0.05 < P<0.1). Ageing had a significant inverse relationship with arterial distensibility in normal subjects (P < 0.05), but not in heart failure patients (P = 0.59). No subject in the normal group had an arterial distensibility lower than 0.1 % per mmHg, in comparison with 28 % (17/60) in the heart failure group. Peripheral arterial distensibility has been shown to be significantly lower in heart failure patients in comparison with normal subjects. High SBP and low LVEF were the main factors associated with low arterial distensibility in heart failure patients.     

Effect of recombinant human erythropoietin on the rate of Na+,H+-exchange in erythrocytes from patients with chronic renal failure on hemodialysis

The study explores the effect of recombinant human erythropoietin and the rate of Na⁺,H⁺-exchange in erythrocytes from patients with chronic renal failure undergoing hemodialysis. The rate of Na⁺,H⁺-exchange in erythrocytes from patients was higher than in the control and remained unchanged after 24 months of treatment with erythropoietin. Therapy with recombinant human erythropoietin does not normalize the Na⁺,H⁺-exchange mechanism. It is concluded that factors underlying disturbances of ion transport in erythrocytes from uremic patients cannot be corrected with erythropoietin. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 12, pp. 613–615, December, 1997     

Cystine transport and glutathione metabolism in human erythrocytes

Cystine was transported into human erythrocytes in the presence of tertiary-butyl hydroperoxide (t-BH) or 1-chloro-2, 4-dinitrobenzene (CDNB). The transport rate of cystine was dependent on the extracellular concentration of t-BH or CDNB, and on the incubation time. By Dowex-1 column chromatography, the transported cystine was incorporated into fractions of glutathione disulfide (GSSG) and glutathione-S (GSH-S) conjugate. Cystine was also transported into reconstituted erythrocyte ghost with GSSG. The transport of cystine was Na+ dependent and decreased in the presence of N-ethylmaleimide, and it was competitively inhibited by DL-homocystine and L-alanine. The inhibition rates by DL-homocystine and L-alanine were 75% and 68%, with similar Ki values of 0.7 mM and 0.6 mM, respectively. The Km value for cystine transport was 0.15 mM. The activity of GSH-cystine transhydrogenase was detected in the hemolysate and this enzyme is thought to catalyze the action of incorporation of cystine into GSH. This enzyme was partially purified from normal human erythrocytes. In the presence of CDNB, similar rates of cystine transport were observed among the diabetic patients (n = 11), hypoxemic patients (n = 10) and the control subjects (n = 20). It is suggested that cystine transport is induced for glutathione synthesis when human erythrocytes are exposed to oxidative stresses.     

Stoichiometry of sodium and potassium transport in erythrocytes from patients with myotonic muscular dystrophy.

1. 22Na and 42K radioisotopes were used to measure Na efflux and K influx in identical suspensions of fresh erythrocytes from myotonic dystrophy patients and matched controls under the same conditions and in the same time interval. K was present in concentration 10 mM in the suspending medium to prevent Na-for-Na exchange. Each flux was measured in the presence and absence of ouabain. The mean ouabain-sensitive Na efflux rate in controls (2-33+/-0-13, S.E. of mean, m-equiv/1. cells.hr) was significantly greater (P less than 0-001) than the corresponding rate in myotonic dystrophy (1-64+/-0-09). 2. No significant differences between myotonic dystrophy and controls in mean ouabain-insensitive Na efflux, mean ouabain-sensitive K influx, or mean ouabain-insensitive K influx were found. 3. The stoichiometric ratio (ouabain-sensitive Na efflux)/(ouabain-sensitive K influx) was determined for each flux experiment. The mean stoichiometric ratio determined in controls (1-46+/-0-08) reconfirms extensive previous evidence favouring a 3Na-for-2K active exchange in controls. 4. The mean stoichiometric ratio determined in myotonic dystrophy (1-01+/-0-06) is statistically significantly different (P less than 0-001) from that in controls. These findings are interpreted as indication of 2Na-for-2K exchange in erythrocytes from patients with myotonic dystrophy.     

Effects of theophylline on erythropoietin production in normal subjects and in patients with erythrocytosis after renal transplantation

BACKGROUND. Erythrocytosis occurs in 10 to 15 percent of renal-transplant recipients, and there is in vitro evidence that the production of erythropoietin is modulated by adenosine. METHODS. We prospectively evaluated the effects of theophylline, a nonselective adenosine antagonist, in eight patients with erythrocytosis after renal transplantation and in five normal controls. RESULTS. After an eight-week course of theophylline treatment, the mean (+/- SEM) serum erythropoietin levels were significantly reduced in both the renal-transplant recipients (from 60 +/- 14 units per liter at base line to 9 +/- 7 units after treatment; P less than 0.05) and the normal subjects (from 6.9 +/- 0.8 units per liter at base line to 4.7 +/- 0.5 units per liter after treatment; P less than 0.05). Similarly, the hematocrits were reduced in both the transplant recipients (from 0.58 +/- 0.04 at base line to 0.46 +/- 0.03 after treatment; P less than 0.05) and the normal subjects (from 0.43 +/- 0.01 at base line to 0.39 +/- 0.01; P less than 0.05). In the renal-transplant recipients, red-cell mass was also reduced after eight weeks of theophylline (from 3197 +/- 82 ml at base line to 2273 +/- 69 ml after treatment; P less than 0.05). The previous requirement of weekly phlebotomy was eliminated in all recipients. Plasma and urinary cyclic AMP levels were not increased. These effects were reproducible when the subjects were rechallenged with theophylline after a recovery period. CONCLUSIONS. Theophylline attenuates the production of erythropoietin in both normal subjects and patients with erythrocytosis after renal transplantation and may be useful in the treatment of the latter condition.     

The rate of loss of CR1 from ageing erythrocytes in vivo in normal subjects and SLE patients: no correlation with structural or numerical polymorphisms.

The stability of CR1 (complement receptor type 1) on ageing erythrocytes in vivo was examined in a group of normal subjects who had been genotyped using a restriction fragment length polymorphism (detected using a cDNA probe for CR1) that correlates with the numerical expression of CR1 on normal erythrocytes (H = allele correlating with high expression, L = low). Erythrocytes were separated into 5 fractions of increasing age on discontinuous Percoll gradients. Mean CR1 numbers on erythrocytes fell from 636 molecules per cell in the first fraction to 384 in the fifth in the HH group and from 478 to 315 in the LL group. There was no difference in the rate of decline of CR1 numbers between the groups. A group of nine SLE patients was also studied in the same way; their genotypes were HH (four) and HL (five). Mean CR1 numbers amongst all of these patients fell from 477 to 232, a faster rate of decline than in a genotypically matched group of normal subjects. There was no difference in the prevalence of the different structural allotypes amongst 30 SLE patients compared with 21 normal subjects. These data provide further evidence that there are enhanced extracellular mechanisms for the removal of CR1 from erythrocytes of SLE patients and do not support the hypothesis that inherited variation in CR1 expression on erythrocytes increases disease susceptibility to SLE.     

Surface markers on human lymphocytes: studies of normal subjects and of patients with primary immunodeficiencies

Peripheral blood lymphocytes of twenty normal controls and twelve patients with primary immunodeficiencies were examined for surface membrane Ig and receptors for C3 complement (B cell markers) and for spontaneous rosette formation with sheep erythrocytes (T cell markers). In patients with defects in T cell function no lymphocytes forming spontaneous rosettes were seen. In patients with B cell deficiency they were normal or increased. Lymphocytes with membrane immunoglobulins were normal in patients with T cell defect and absent in patients with severe agammaglobulinaemia. Lymphocytes with receptors for C3 complement were increased in patients with T defect and normal in patients with most other forms of immunodeficiency studied.     

Kinetics of elimination of thiocyanate in 7 healthy subjects and in 8 subjects with renal failure

Summary The concentration of thiocyanate in the serum of eight test subjects with renal failure and seven healthy control subjects was measured, as it declined with time, after oral doses of thiocyanate or i.v. injections of nitroprusside had been administered. Additional measurements were taken, on the healthy subjects only, of the concentrations of thiocyanate in the urine, and also of the influence of an increased chloride intake on the rate of elimination of thiocyanate. For the healthy subjects an elimination half-life of between one and five days (mean c. 3 days) was found. Increasing the chloride elimination rate to approximately twice normal did not significantly speed up the rate of thiocyanate elimination. The amounts of thiocyanate which had been administered as doses reappeared almost exclusively in the urine. For the subjects with renal failure, the elimination half-life had a mean value of approximately nine days. The elimination constants were found to be proportional to the creatinine-clearance rates. Thek _e value at a creatinine-clearance of zero ml/min was approximately 15% of thek _e value at a creatinine-clearance rate of 120 ml/min. The distribution volumes for thiocyanate were greater for the patients with renal failure than for the healthy subjects. The conclusions for therapies using nitroprusside are discussed.     

Lead transport and binding by human erythrocytes in vitro

Transport and binding of Pb²⁺ by human erythrocytes were examined for cell Pb contents in the 1–10 M range, using the ²⁰³Pb isotope. Pb²⁺ crosses the erythrocyte membrane by the anion exchanger, and can also leave erythrocytes by a vanadate-sensitive pathway, identified with the Ca²⁺ pump. However, Pb²⁺ exit is very much less than expected from earlier experiments with resealed erythrocyte ghosts [Simons TJB (1988) J Physiol (Lond) 405:105–113] and the distribution of Pb²⁺ across the erythrocyte membrane is close to equilibrium. The high ratio of erythrocyte to plasma Pb seen in vivo appears to be due to the presence of a labile Pb²⁺-binding component present in erythrocyte cytoplasm.     

Glucose metabolism in non-diabetic and insulin-dependent diabetic subjects with end-stage renal failure

Chronic uremia is frequently associated with an impaired carbohydrate tolerance. During the past decade considerable progress have been made in characterizing and quantifying this biochemical abnormality in end-stage renal failure (ESRF). Primarily, this has been possible by means of the glucose clamp technique which basically makes it possible to evaluate insulin sensitivity and glucose-stimulated insulin secretion. Combined with the use of tracer dilution technique, hepatic vein catheterization technique, infusion of somatostatin, forearm or leg techniques and indirect calorimetry, insight into several other major parameters of glucose kinetics has been achieved; i.e. insulin-mediated glucose uptake (IMGU), glucose-induced glucose uptake (GIGU), hepatic glucose production (HGP) splanchnic glucose uptake and oxidative and nonoxidative glucose disposal. Of course, these extra facets make the clamp procedure less feasible to accomplish for technical reasons and demand an extensive knowledge of the limitations of these methods. One major factor behind the reduced glucose tolerance in uremia is an impaired sensitivity to insulin (insulin resistance) in peripheral tissues, mainly in skeletal muscle. In non-dialysed uremic patients the insulin dose-response curve is characterized by a decreased maximal response and by a rightward shift. In general, the insulin resistance is pronounced, but a few weeks on maintenance hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) are enough to improve insulin action significantly. Occasionally, IMGU has been found normal in patients on long-term HD. In contrast to insulin-stimulated glucose uptake, basal glucose turnover is normal in patients with ESRF. The ability of glucose to enhance its own uptake is difficult to measure in human studies, because even small amounts of insulin is able to modulate GIGU profoundly. At basal insulinemia, however, GIGU is markedly impaired in uremia. Recently, it has been suggested that the uremic insulin resistance is located not only in peripheral tissues but also in the liver. At low insulin concentrations, the restraining potency of insulin on HGP seems to be decreased in uremia. Splanchnic glucose uptake is hardly affected, but is always very insensitive to insulin. The glucoregulatory function of the liver is further disturbed in uremia. Acute glucagon exposure elicits an inadequate glucose release, suggesting a coexisting resistance to glucagon. In vitro studies have shown, that the first step in the cascade of reactions initiated by insulin, namely binding to its specific receptor is normal in uremia. In addition, the activity of key enzymes such as the insulin receptor kinase and glycogen synthase have been found within normal in the uremic muscle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytogenetically marked clones in human fibroblasts cultured from normal subjects

Clones of cytogenetically abnormal cells have been recognized in fibroblasts cultured from normal human adult skin. No such clones have been observed in human embryo skin fibroblasts cultured in the same way. Although the culture conditions may have played some part in the emergence of these clones, it is possible that the abnormal cells from which the clones were derived were present in vivo.     

The effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.