Thyroid-stimulating hormone (TSH) binding to extrathyroidal human tissues: TSH binding to extrathyroidal human tissues: TSH and thyroid-stimulating immunoglobulin effects on adenosine 3',5'-monophosphate in testicular and adrenal tissues. Binding of [125I]bovine TSH to human thyroid, testicular, fat, adrenal, liver, kidney, pancreas, and lung cell membranes has been studied. The first four tissues were found to have comparable high affinity constant values; the rest of the tissues lacked high affinity sites. With the exception of fat tissue, the capacities of the high affinity sites of the first four tissues were similar. Bovine TSH concentrations of 100-20,000 microIU/ml stimulated increased cAMP production in human cryopreserved testicular slices. Forty percent of the specimens of thyroid-stimulating immunoglobulin (TSI) from Graves' disease sera also increased human testicular cAMP production. In addition, bovine TSH caused a significant rise in cAMP in the whole decapsulated rat testis. Twenty-five percent of the TSI specimens tested also induced such responses. The rat adrenal gland responded with increased cAMP production to concentrations of 1,000 microU/ml bovine TSH. The physiological significance of high affinity bovine TSH and TSI binding and subsequent cAMP production in nonthyroidal tissues in not known. However, since these stimulators are present in hypothyroidism and hyperthyroidism, respectively, it is possible that the pathophysiological effects of this binding could be of some importance.


The role of serum lipoproteins in steroidogenesis by the human fetal adrenal cortex. In the present investigation it was found that human fetal adrenal tissue maintained in organ culture secreted appreciable quantities of dehydroisoandrosterone sulfate (DS) and cortisol. Pregnenolone was also secreted in significant amounts, principally as the sulfate ester. The highest rate of secretion of these steroids by fetal adrenal tissue occurred when both ACTH and whole human serum were present in the culture medium. In the absence of ACTH, steroid secretion was low. When whole serum was replaced by lipoprotein-poor serum, the steroidogenic response to ACTH was markedly attenuated but not abolished. On the basis of these findings, it is concluded (1) that the human fetal adrenal can synthesize steroid hormones de novo from cholesterol, (2) that ACTH is an important stimulant of steroidogenesis by the human fetal adrenal, and (3) that plasma lipoproteins are a major source of the cholesterol utilized by the human fetal adrenal for steroidogenesis. Hence, it is likely that factors which regulate the production of fetal plasma lipoproteins are important determinants of fetal adrenal steroidogenic activity.


Adrenocortical function, electrolyte metabolism, and blood pressure during prolonged adrenocorticotropin infusion in juvenile hypertension. The effect of a continuous 5-day ACTH infusion (40 U/24 h) on adrenocorticoid function, electrolyte metabolism, and blood pressure was investigated in eight normotensive children and eight patients with hypertension of unknown origin. There was a continuous rise of plasma cortisol and deoxycorticosterone in all patients. Plasma aldosterone rose transiently in the normotensive and the hypertensive group. A transient kaliuresis and a continuous fall in serum K+ were observed in all patients. ACTH induced sodium retention and weight gain. The observed increase in systolic blood pressure correlated significantly with the cumulative sodium retention in the normotensive and the hypertensive groups. No correlation between sodium retention and diastolic pressure was found. ACTH on a low salt diet (10 meq/24 h) produced a blood pressure rise which was smaller than that on regular salt. The blood pressure rise did not correlate with any of the hormones measured. This study provides evidence for an unidentified ACTH-stimulable adrenal factor capable of raising blood pressure in normotensive children and patients with juvenile hypertension. The ACTH-induced blood pressure rise is only partly salt dependent and the mechanism of the rise remains unclear.


Effects of nicotinic acid therapy on plasma high density lipoprotein subfraction distribution and composition and on apolipoprotein A metabolism. This report describes the effects of pharmacologic doses (3 g/d) of nicotinic acid on the plasma distribution and chemical composition of the high density lipoprotein (HDL) subfractions HDL(2) and HDL(3) and examines the influence of the drug on the metabolism of the major HDL apoproteins, apolipoproteins A-I (ApoA-I) and A-II (Apo-II). The drug lowered plasma cholesterol (15%, P < 0.05) and triglyceride (27%, P < 0.01); the former effect a result of a fall in the amount of cholesterol associated with very low density lipoproteins (31%, P < 0.02) and low density lipoproteins (36%, P < 0.02). Conversely, it raised plasma HDL cholesterol (23%, P < 0.05) and increased (by 345%) the plasma HDL(2):HDL(3) ratio. The latter derived from an absolute increment (646%) in circulating HDL(2), coupled with a fall (47%) in HDL(3). This change was not associated with major alterations in the overall cholesterol (free and esterified), triglyceride, phospholipid, or protein content of the subfractions; however, it was accompanied by substantial changes in their protein composition. In particular, the molar ratio of ApoA-I:ApoA-II in HDL(3) declined from 2.7:1 to 2.1:1 during nicotinic acid treatment.Significant perturbations of ApoA-I and ApoA-II metabolism accompanied the drug-induced HDL subfraction redistribution. Specifically, the plasma concentration of ApoA-I rose by 7% (P < 0.05) because of a decrease in its fractional catabolic rate. Moreover, whereas before treatment 6 and 94% of the plasma ApoA-I circulated with HDL(2) and HDL(3), after commencement of nicotinic acid therapy this distribution became 49 and 51% in HDL(2) and HDL(3), respectively. ApoA-II was found mainly in HDL(3), both before and during nicotinic acid treatment. Administration of the drug caused a 14% reduction in its plasma concentration (P < 0.05), which derived principally from a fall (22%, P < 0.01) in its synthetic rate. These data suggest that the effects of nicotinic acid on the HDL subfraction distribution may be mediated via (a) net transfer of ApoA-I from HDL(3) to HDL(2) and (b) a reduction in ApoA-II synthesis. Our present understanding of the association between HDL and atherosclerosis indicates that such changes may have prophylactic value in the prevention of coronary artery disease.


Inhibition of angiotensin converting enzyme activity in cultured endothelial cells by hypoxia. Endothelial cells in tissue culture degrade bradykinin and convert angiotensin I to angiotensin II. These are both functions of a single dipeptidyl hydrolase, angiotensin converting enzyme. Monolayer cultures were prepared from human, rabbit, pig, and calf vessels. Angiotensin converting enzyme activity was assessed by adding either bradykinin or angiotensin I to the cells in culture flasks, and measuring residual peptide over time by radioimmunoassay. Peptide degradation was inhibited by the specific converting enzyme inhibitor, SQ 20881. The flasks were equilibrated with varying hypoxic gas mixtures: hypoxia rapidly (less than 2 min) decreased enzyme activity and room air restored it as rapidly. The extent to which activity was reduced was a direct function of PO2 (r = 0.93, P less than 0.001), and there was no enzyme activity below a PO2 of 30 mm Hg. Four preparations were studied with respect to decrease in enzyme activity by hypoxia: (a) intact cells in monolayer, (b) sonicated cells, (c) sonicated cells from which converting enzyme was partially dissolved by a detergent, and (d) purified converting enzyme. Hypoxia had progressively less of an inhibiting effect on the enzyme activity of the preparations as the degree of cell integrity decreased. Hypoxia inhibits angiotensin converting enzyme activity in cultured endothelial cells, but the effect of hypoxia is not on the enzyme per se, but appears to be a unique characteristic of the endothelial cell.


Effects of glucose and parathyroid hormone on the renal handling of myoinositol by isolated perfused dog kidneys. The effects of glucose and parathyroid hormone (PTH) on the transport and metabolism of myoinositol (MI) and [2-(3)H]MI were studied in isolated perfused dog kidneys. Studies during perfusion of kidneys with normal and elevated glucose concentrations demonstrated that under normal conditions the isolated kidney reabsorbed 94.7+/-0.2% of the filtered MI, and the renal production of (3)H-metabolities of MI was 117.9+/-6% of the filtered MI load. This indicated that entry of MI into tubular cells by reabsorption was not the sole pathway for entry into the pool of MI within the kidney undergoing catabolism. High glucose perfusate decreased MI reabsorption to 68.6+/-4.7% and thus decreased delivery of [2-(3)H]MI into the catabolic pool from the reabsorptive pathway. In the high glucose experiments, the rate of [2-(3)H]MI catabolism exceeded [2-(3)H]MI reabsorption by the same fraction as in normal glucose experiments, which indicates that high glucose did not affect nonreabsorptive access of MI to the catabolic site. In contrast to the effects of glucose, PTH administration resulted in an increase in perfusate MI concentration and a decrease in the perfusate [2-(3)H]MI specific activity. Concomitantly, urinary MI and [2-(3)H]MI concentrations were increased, again with a decrease in [2-(3)H]MI specific activity. These results indicate that PTH caused a release of MI into the urine (not the same as decreased MI reabsorption, which would not affect urinary [(3)H]MI specific activity) and into the perfusate of the isolated kidneys. These effects on MI release were about coincidental with the increase in urinary cyclic 3',5'-AMP after PTH and preceded the peak phosphaturic effect of PTH. There was no detectable effect of PTH on MI synthesis from glucose as a source of the MI released into the urine and perfusate. However, PTH temporarily halted accumulation of tritiated MI catabolites. There was no effect of inactivated PTH on urinary cyclic 3',5'-AMP or on MI transport, which indicates that the PTH effect on MI handling was a specific hormonal effect. These studies clarify the renal metabolism of MI, and they demonstrate heretofore unknown effects of PTH on the renal handling and metabolism of MI. The effects of PTH on renal MI metabolism have important implications in renal carbohydrate metabolism and phospholipid turnover.


Adenylate cyclase in thymus-derived and bone marrow-derived lymphocytes from normal donors and patients with chronic lymphocytic leukemia. Lymphocytes were purified from peripheral blood of normal donors and patients with chronic lymphocytic leukemia (CLL) by Ficoll-Hypaque centrifugation. Adenylate cyclase activity, expressed as picomoles [(32)P]cyclic AMP generated per milligram protein per minute, was 57+/-4 in normals and 26+/-4 in CLL patients. Enzyme activity, expressed as picomoles [(32)P]cyclic AMP generated per 10(6) lymphocytes per minute, was 2.09+/-0.19 for normal lymphocytes and 1.10+/-0.16 for CLL lymphocytes. The differences between normal and CLL peripheral lymphocytes are highly significant (P < 0.001) with either method of calculating activity. Cyclic AMP levels (picomoles per 10(6) lymphocytes) also differed significantly: 1.38+/-0.29 for normals and 0.45+/-0.08 for CLL lymphocytes. Adenylate cyclase was assayed in lymphocytes enriched for bone marrow-derived (B) cells by removing E-rosetted thymus-derived (T) cells, and enriched for T cells by harvesting E-rosetted lymphocytes or by removing B cells with nylon wool absorption. Solutions to simultaneous equations gave the following calculated enzyme activities for pure B- and T-cell subpopulations (in picomoles [(32)P]cyclic AMP generated per milligram mg protein per minute): normal B, 196+/-22; normal T, 30+/-10; CLL B, 34+/-6; CLL T, 19+/-4. Thus. normal B-lymphocyte adenylate cyclase exceeds normal T-lymphocyte activity by more than sixfold, whereas in the case of CLL the enzyme activity in B lymphocytes is markedly reduced to levels comparable to T lymphocytes. The responses of lymphocytes to stimulation with the hormones prostaglandin E(1) and isoproterenol, and with NaF, were assessed. Compared with normal lymphocytes, enzyme activities were reduced in CLL lymphocytes incubated with these agents, but to a degree paralleling the reduced basal activities. Thus, the ratios between stimulated and basal adenylate cyclase levels in Ficoll-Hypaque-purified, normal lymphocytes were 2.3+/-0.1 after incubation with 10 muM isoproterenol, and 3.9+/-0.2 with 10 mM NaF, values which did not differ significantly from those obtained with CLL lymphocytes. When the enzyme activities calculated for purified T- and B-lymphocyte subpopulations were used to derive the stimulation ratios, the responses of normal and CLL T and B cells to these agents were also indistinguishable. The simplest explanation for these findings is a reduced number of normally responsive enzyme sites on the surface membranes of CLL lymphocytes, although alternative explanations are possible.


Evidence for an intrinsic renal tubular defect in mice with genetic hypophosphatemic rickets. To investigate the role of parathyroid hormone (PTH) and(or) an intrinsic renal tubular reabsorptive defect for phosphate in mice with hereditary hypophosphatemic rickets, we performed clearance and micropuncture studies in hypophosphatemic mutants and nonaffected littermate controls. Increased fractional excretion of phosphate in mutants (47.2+/-4 vs. 30.8+/-2% in controls) was associated with reduced fractional and absolute reabsorption in the proximal convoluted tubule and more distal sites. Acute thyropara-thyroidectomy (TPTX) increased phosphate reabsorption in both mutants and controls with a fall in fractional phosphate excretion to congruent with7.5% in both groups indicating that PTH modified the degree of phosphaturia in the intact mutants. Absolute reabsorption in the proximal tubule and beyond remained reduced in the mutants, however, possibly because of the reduced filtered load. Serum PTH levels were the same in intact mutants and normals as was renal cortical adenylate cyclase activity both before and after PTH stimulation. To evaluate the possibility that the phosphate wasting was caused by an intrinsic tubular defect that was masked by TPTX, glomerular fluid phosphate concentration was raised by phosphate infusion in TPTX mutants to levels approaching those of control mice. Phosphate excretion rose markedly and fractional reabsorption fell, but there was no change in absolute phosphate reabsorption in either the proximal tubule or beyond, indicating a persistent reabsorptive defect in the absence of PTH. We conclude that hereditary hypophosphatemia in the mouse is associated with a renal tubular defect in phosphate reabsorption, which is independent of PTH and therefore represents a specific intrinsic abnormality of phosphate transport.


Relationship of receptor affinity to the modulation of thyroid hormone nuclear receptor levels and growth hormone synthesis by L-triiodothyronine and iodothyronine analogues in cultured GH1 cells. We have previously demonstrated that L-triiodothyronine (L-T3) induces an increase in growth hormone synthesis and messenger RNA in cultured GH1 cells, a rat pituitary cell line. In addition to regulating the growth hormone response, L-T3 elicits a time- and dose-dependent reduction in the level of its nuclear receptor, which is a direct function of the occupancy of the receptor binding site. In this study we have compared the relative affinity of L-T3, triiodothyroacetic acid, D-triiodothyronine (D-T3), and L-thyroxine (L-T4) for the receptor with the induction of the growth hormone synthesis and the ability of these compounds to elicit a reduction in thyroid hormone nuclear receptor levels. Triiodothyroacetic acid and D-T3 were specifically examined because the biologic effect of these compounds in the intact rat is significantly lower than predicted by their affinity for the receptor using isolated rat liver nuclei in vitro. In intact cells each compound demonstrated an excellent relationship between the relative receptor affinity, the induction of growth hormone production, and the concentration-dependent reduction in nuclear receptor levels. With the exception of D-T3, the relative affinity of iodothyronine was identical for the receptor using intact cells in serum-free media, or isolated GH1 cell nuclei in vitro. The apparent receptor affinity of D-T3 with intact cells was 5.5-fold lower than with isolated nuclei, which suggests a decrease in cell entry of D-T3 relative to the other iodothyronines. Quantitation of the [125I]iodothyronine associated with the receptor in GH1 cells after a 36-h incubation with L-125I-T4 was 90% L-T4 and 10% L-T3, which indicates that the major effect of L-T4 in GH1 cells is a result of intrinsic L-T4 activity. Studies with dispersed rat anterior pituitary cells demonstrated that L-T3 induces growth hormone synthesis and elicits a reduction in nuclear receptor levels in the same fashion as GH1 cells. The observation that thyroid hormone influences dispersed rat pituitary cells in a fashion qualitatively similar to GH1 cells may have implications for the growth hormone response of the somatotroph cell in vivo to different thyroidal states.


Kinetic model for production and metabolism of very low density lipoprotein triglycerides. Evidence for a slow production pathway and results for normolipidemic subjects. A model for the synthesis and degradation of very low density lipoprotein triglyceride (VLDL-TG) in man is proposed to explain plasma VLDL-TG radioactivity data from studies conducted over a 48-h interval after injection of glycerol labeled with 14C, 3H, or both. The curve describing the radioactivity of plasma VLDL triglycerides reaches a maximum at about 2 h, after which the decay is biphasic in all cases; the late curvature becoming evident only after 8--12 h. To fit the complex curve, it was necessary to postulate two pathways for the incorporation of plasma glycerol into VLDL-TG, one much slower than the other. A process of stepwise delipidation of VLDL in the plasma compartment, previously proposed for VLDL apoprotein models, was also necessary. Predicted VLDL-TG synthesis rates calculated with this model can differ significantly from those based on experiments of shorter duration in which the slow VLDL-TG component is not apparent. The results of these studies strongly support the interpretation that the late, slow component of the VLDL-TG activity curve is predominantly due to the slowly turning-over precursor compartment in the conversion pathway and is not due either to a slow compartment in the labeled precursor, plasma free glycerol, or to an exchange of plasma VLDL-TG with an extravascular compartment. It also cannot, in these studies, be attributed to a slowly turning-over VLDL-TG moiety in the plasma. The model was tested with data from 59 studies including normal subjects and patients with obesity and(or) various forms of hyperlipoproteinemia. Good fits were obtained in all cases, and the estimated parameter values and their uncertainties for 13 normolipemic nonobese subjects are presented. Sensitivty testing was carried out to determine how critical various parameter estimations are to the assumptions introduced in the modeling.


Transport of very low density lipoprotein triglycerides in varying degrees of obesity and hypertriglyceridemia. Measurements of transport of triglycerides (TG) in very low density lipoproteins (VLDL) were carried out in 59 patients by injection of radioactive glycerol, determinations of specific activities of VLDL-TG for 48 h thereafter, and treatment of the data by multicompartmental analysis. The patients were divided into three groups: normal weight (89-120% ideal weight), mildly obese (120-135% ideal weight), and markedly obese (135% ideal weight). They had varying levels of VLDL-TG ranging from normal to markedly elevated. In many subjects, there was a positive correlation between concentrations and transport of VLDL indicating that overproduction of VLDL-TG contributed to hypertriglyceridemia. In others, and particularly in several markedly obese subjects, transport rates were greatly increased without significant hypertriglyceridemia, suggesting that they had enhanced capacity to clear TG. In all groups, however, there were patients whose degree of hypertriglyceridemia seemed out of proportion to their transport rates. This finding and the fact that many patients have increased secretion of VLDL-TG without elevated plasma TG suggests that both overproduction of VLDL-TG and insufficient enhancement of clearance contributed to the development of hypertriglyceridemia.The data showed a poor correlation between transport rates determined by our multicompartment analysis and single-exponential analysis used previously by other investigators (r = 0.46); this comparison was not improved by segregating patients according to their degree of obesity. Although two conversion pathways (fast and slow synthetic paths) were required to fit the data, there was no correlation between transport rates and the ratio of the two pathways. Also, despite the known pathway of conversion of VLDL to low density lipoprotein, no correlation was found between VLDL-TG transport rates and estimated low density lipoprotein-cholesterol concentrations.


Distribution of beta-endorphin immunoreactivity in normal human pituitary. Recent immunohistochemical demonstration of calcitonin in rat pituitary has suggested that calcitonin, in addition to ACTH, endorphins, lipotropins, and melanocyte-stimulating hormones might be derived from a 31,000-dalton glycoprotein percursor molecule. This immunoperoxidase study demonstrates a similar distribution for beta-endorphin and ACTH immunoreactivity in human pituitary; however, the two peptides are not necessarily present in the same cells at all times. Calcitonin could not be demonstrated in human pituitary under conditions suitable for demonstration of the peptide in thyroid C cells. Weakly positive immunostaining could be obtained only with much increase in antiserum concentration and length of incubation, and higher concentrations of calcitonin were needed to abolish staining in preabsorption studies. It thus appears that the immunoreactive calcitonin in human pituitary differs from that in thyroid C cells. Likewise, we could not demonstrate immunoreactive endorphin in any developmental stage of medullary thyroid carcinoma. Our study suggests that caution should be applied in considering a physiologic role for calcitonin in the pituitary and in postulating a common peptide origin for endorphin and calcitonin in humans.


Positive rate-sensitive corticosteroid feedback mechanism of ACTH secretion in Cushing's disease. To define the nature of the disturbance of the corticosteroid feedback mechanism in Cushing's disease, the dynamic aspects of the ACTH response to corticosteroid administration have been studied in patients with Cushing's disease after total adrenalectomy (C.d. post adx.). The results were compared with those obtained in patients with Addison's disease (control group). Different experimental designs for administration of cortisol were chosen to provide extreme variations in the input signal. The response of the system was evaluated by measuring plasma ACTH concentrations (radioimmunoassay) at short time intervals. Infusion of cortisol at constant rate (50 mg/h for 2 h) resulted in a transient, paradoxical rise in ACTH levels with a maximum at 15 min. (315+/-65%, mean+/-SEM). In contrast, in the control group there was an immediate and rapid decrease in ACTH levels with a significant inhibition after 15 min (80+/-6%, mean+/-SEM). Infusion of 50 mg cortisol for 5 and 15 min, respectively, produced an increase in ACTH levels, which was confined to the time when cortisol levels were rising (maximum: 137+/-30% and 139+/-10% at 5 and 15 min, respectively, mean+/-SEM). This increase corresponded in time to the first decrease in ACTH levels in the Addisonian patients. With bolus injections of 25 mg cortisol, ACTH levels remained unchanged during the first 15 min. The time-course in the patients with C.d. post adx. was essentially the same as in the Addisonian patients. From these results it is concluded that in the patients with C.d. post adx. the rapid, rate-sensitive feedback mechanism was converted into a positive one, whereas the delayed, dose-sensitive mechanism was completely undisturbed. The capacity of dexamethasone to activate rate-sensitive feedback elements was markedly diminished. Accordingly, there were only minor positive feedback effects upon ACTH secretion in the patients with C.d. post adx.


Membrane fluidity in human and mouse Chediak-Higashi leukocytes. Polymorphonuclear leukocytes from humans and mice with the Chediak-Higashi syndrome were characterized by spin label electron spin resonance spectrometry. Our results suggest that cells from afflicted mice and humans have membranes more fluid than controls. Order parameters for a spin label that probes near the membrane surface were 0.652 for normals and 0.645 for two Chediak-Higashi patients. Cells from Chediak-Higashi mice showed similar differences, as did isolated plasma membrane fractions. An increased membrane fluidity was also detected with a spin label that probes deeper in the bilayer. In vitro treatment of Chediak-Higashi mouse cells with 0.01 M ascorbate increased the order parameter to normal levels. In vitro incubation of mouse Chediak-Higashi cells with glucose oxidase increased the order parameter, similar to the effect of ascorbate. This increase was abolished when catalase was added to the incubation medium. In vitro incubation with dibutyryl cyclic guanosine monophosphate (1 muM to 0.1 mM) did not normalize order parameters. These results indicate that fluidity of Chediak-Higashi cell membranes was affected by treatments expected to alter the oxidation: reduction potential of the environment but was not affected by treatments expected to alter the ratio of intracellular cyclic nucleotides. The latter treatment would affect microtubule assembly. Therefore, it appears that the membrane fluidity abnormalities as demonstrated by electron spin resonance and the earlier demonstrated microtubule dysfunctions characteristic of Chediak-Higashi cells are coexisting defects and are probably not directly related.


Quantitation of the transfer of surface phospholipid of chylomicrons to the high density lipoprotein fraction during the catabolism of chylomicrons in the rat. Small chylomicrons (CM) labeled with cholesterol, cholesterol ester, phospholipid, and, in some cases, protein, were used to study the fate of these constituents as the CM are catabolized in the circulations of the hepatectomized and intact rat. In the hepatectomized animal after (1/2) h, CM are greatly reduced in volume, surface area, and diameter. During this period, the CM lost >92% of the mass of their triacylglycerol, >77% of the mass of their phospholipid, and >39% of their protein. Compared to the injected CM, the chemically altered particles, called CM "remnants," have a reduction in volume of 96% and in surface area of 88%. The labeled cholesterol esters remain with the CM remnants but, strikingly, a major fraction of the labeled phospholipids and labeled soluble apoproteins leave the CM and are found in the high density lipoprotein (HDL) fraction. The chemical composition of this HDL fraction contains relatively more phospholipid and less cholesterol ester than normal rat HDL. Because of the difference in composition of HDL between normal rats and those given CM, we estimate that the HDL phospholipid pool increased by congruent with25% by the infusion of congruent with 4-5 mg of CM phospholipid. Approximately 5 mg of phospholipid is secreted on CM by a fed rat in 1 h. The findings in hepatectomized rats indicate that a major fraction of the phospholipid and a minor fraction of the protein (soluble non-B apoproteins) of newly secreted CM are transferred from the CM to the HDL fraction during remnant formation. The same process probably occurs in intact rats except that the remnant particles are rapidly removed from the plasma by the liver and a smaller fraction of the surface of the CM enters the HDL fraction.


Modulation of neutrophil function by lysozyme. Potential negative feedback system of inflammation. Host responses to infectious organisms should be modulated so that tissue-damaging products of inflammatory cells do not produce excessive destruction of normal tissue. Lysozyme, which is continuously secreted by monocytes, which, in turn, migrate relatively late to inflammatory areas, was found to significantly dampen several responses of neutrophils to inflammatory stimulants. Thus, human lysozyme obtained and purified from the urine of patients with monocytic leukemia (but not its structurally similar and comparably cationic analogue, eggwhite lysozyme) depresses chemotaxis of normal neutrophils to activated complement, bacterial supernate, and N-formylmethionyl-phenylalanine. In addition, human (but not eggwhite) lysozyme depresses oxidative metabolism (hexose monophosphate shunt activity) and superoxide generation of neutrophils. The specificity of the suppressive effects was indicated by inhibition studies with rabbit antihuman lysozyme antibody, and with the trisaccharide of N-acetylglucosamine, a specific inhibitor of lysozyme. The results suggest that lysozyme, a product of inflammatory cells themselves, may function in a negative feedback system to modulate the inflammatory response.


Human intestinal lipoproteins. Studies in chyluric subjects. To explore the role of the human intestine as a source of apolipoproteins, we have studied intestinal lipoproteins and apoprotein secretion in two subjects with chyluria (mesenteric lymphatic-urinary fistulae). After oral corn oil, apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II) output in urine increased in parallel to urinary triglyceride. One subject, on two occasions, after 40 g of corn oil, excreted 8.4 and 8.6 g of triglyceride together with 196 and 199 mg apoA-I and on one occasion, 56 mg apoA-II. The other subject, after 40 g corn oil, excreted 0.3 g triglyceride and 17.5 mg apoA-I, and, after 100 g of corn oil, excreted 44.8 mg apoA-I and 5.8 mg apoA-II. 14.5+/-2.1% of apoA-I and 17.7+/-4.3% of apoA-II in chylous urine was in the d < 1.006 fraction (chylomicrons and very low density lipoprotein). Calculations based on the amount of apoA-I and apoA-II excreted on triglyceride-rich lipoproteins revealed that for these lipid loads, intestinal secretion could account for 50 and 33% of the calculated daily synthetic rate of apoA-I and apoA-II, respectively. Similarly, subject 2 excreted 48-70% and 14% of the calculated daily synthetic rate of apoA-I and apoA-II, respectively. Chylous urine contained chylomicrons, very low density lipoproteins and high density lipoproteins, all of which contained apoA-I. Chylomicrons and very low density lipoproteins contained a previously unreported human apoprotein of 46,000 mol wt. We have called this apoprotein apoA-IV because of the similarity of its molecular weight and amino acid composition to rat apoA-IV. In sodium dodecyl sulfate gels, chylomicron apoproteins consisted of apoB 3.4+/-0.7%, apoA-IV 10.0+/-3.3%, apoE 4.4+/-0.3%, apoA-I 15.0+/-1.8%, and apoC and apoA-II 43.3+/-11.3%. Very low density lipoprotein contained more apoB and apoA-IV and less apoC than chylomicrons. Ouchterlony immunodiffusion of chylomicron apoproteins revealed the presence of apoC-I, apoC-II, and apoC-III. In contrast, plasma chylomicrons isolated during a nonchyluric phase revealed a markedly altered chylomicron apoprotein pattern when compared with urinary chylomicrons. The major apoproteins in plasma chylomicrons were apoB, apoE, and the C peptides: no apoA-I or apoA-IV were present in sodium dodecyl sulfate gels indicating that major changes in chylomicron apoproteins occur during chylomicron metabolism. When incubated in vitro with plasma, urinary chylomicrons lost apoA-I and apoA-IV and gained apoE and apoC. Loss of apoA-I and apoA-IV was dependent upon the concentration of high density lipoproteins in the incubation mixture. These studies demonstrate that the human intestine secretes significant amounts of apoA-I and apoA-II during lipid absorption. Subsequent transfer of apoproteins from triglyceride-rich lipoproteins to other plasma lipoproteins may represent a mechanism whereby the intestine contributes to plasma apoprotein levels.


In vitro and in vivo refractoriness to thyrotropin stimulation of iodine organification and thyroid hormone secretion. Earlier studies indicated that initial exposure of thyroid slices to thyrotropin diminished responsiveness of the adenylate cyclase-cyclic AMP system, glucose oxidation, and (32)P(i) incorporation into phospholipids upon readdition of the hormone. The present studies demonstrate that slices from dog, beef, and human thyroid glands initially incubated with thyrotropin (TSH) were less responsive to subsequent addition of the hormone when organification of iodide was examined. Increasing the amount of TSH did not overcome the refractoriness induced by the initial exposure to the hormone. Furthermore, the stimulatory effects of dibutyryl cyclic AMP and prostagladin E(1) were abolished in slices previously incubated with TSH. Development of such refractoriness did not depend upon new protein synthesis and was not abolished by 1 mM prophylthiouracil in the first incubation. Addition of 0.1 muM thyroxine or triiodothyronine or 1.5 muM iodide during all three incubations did not modify the response to TSH, added for the first time in the third incubation. However, 1 mM iodide in the buffer during all three incubations inhibited the response to TSH during the third incubation. During the refractory period, effects of TSH on colloid droplet formation were also diminished. The in vivo effect of TSH on serum l- triiodothyronine in rats was significantly reduced when the rats had been injected with TSH 8 h earlier. These studies demonstrate that TSH-induced refractoriness also includes effects on organification of iodine and secretion of thyroid hormone. The results cannot be adequately explained by unresponsiveness of adenylate cyclase because effects of dibutyryl cyclic AMP and prostagladin E(1) were also inhibited by prior exposure to TSH.


Metabolic studies in an unusual case of asymptomatic familial hypobetalipoproteinemia with hypolphalipoproteinemia and fasting chylomicronemia. A new kindred with asymptomatic hypobetalipoproteinemia is reported. The proband, age 67, differs from previously described cases in several respects: (a) unusually low levels of low density lipoprotein (LDL) cholesterol (4-8 mg/dl); (b) normal triglyceride levels; (c) low levels of high density lipoprotein; (d) mild fat malabsorption; and (e) a defect in chylomicron clearance. On a high-carbohydrate diet his plasma triglyceride levels, instead of rising, actually fell. Turnover of triglycerides in very low density lipoproteins (VLDL) was low (2.8 mg/kg per h). Fractional catabolic rate of LDL protein was just above the normal range (0.655/d) but net turnover was <10% of normal (0.65 mg/kg per d). The half-life of his chylomicrons was 29 min, five times the normal value. Postheparin lipoprotein lipase activity was normal and apolipoprotein C-II, the activator protein for lipoprotein lipase, was present and functional. Apolipoprotein C-III(1), however, was not detected in the VLDL fraction, a finding previously reported in patients with abetalipoproteinemia. Fecal excretion of cholesterol was almost twice normal; total sterol balance was increased by congruent with40%. The unusual features in the proband that distinguish him from previously described cases and from his affected first-degree relatives suggested that, in addition to the basic gene defect affecting LDL metabolism, he might have a second abnormality affecting clearance of chylomicrons and VLDL. The ratio of apolipoprotein E(3) to E(2) in his VLDL fraction was 0.93, just below the lower limit of normal, suggesting heterozygosity for E(3) deficiency. Whether or not this contributes to his hypertriglyceridemia remains to be established.


Production of hybridomas secreting monoclonal antibodies against the lympholine osteoclast activating factor. The human lympholine osteoclast activating factor (OAF) is thought to be involved in several bone-destroying diseases. The current studies were designed to produce monoclonal antibodies against OAF for use in the subsequent design of immunoassays for OAF in clinical samples. Spleen cells from mice immunized with purified human OAF were hybridized with mouse plasmacytoma cells in vitro to yield hybridomas. Several clones of these hybridomas secreted into the culture medium antibodies, which neutralized the biological activity of OAF at dilutions as high as 1:100,000 relative to the initial culture medium. These antibodies did not interfere with the activities of parathyroid hormone in the same systems. These results represent the first report of monoclonal antibodies against a human lympholine, and validate the concept that hybridoma production is a useful technique for developing antibodies against weak or scarce antigens.


Modulation of in vitro erythropoiesis. Studies with euthyroid and hypothyroid dogs. The interactions of  adrenergic agonists and thyroid hormones on the growth of erythroid colony-forming units were studied in cultures of dog marrow before and after the establishment of hypothyroidism. Erythroid colony growth in cultures form euthyroid dogs was enhanced by isoproterenol and other adrenergic agonists having beta 2-receptor specificity. With hypothyroidism, however, this responsiveness was lost, and sensitivity to alpha-agonists, such as phenylephrine and norepinephrine, was acquired. This alteration in receptor specificity appeared to be dependent upon thyroid hormone and was rapidly reversible. Preincubation of marrow cells from hypothyroid animals with thyroid hormone resulted in the reappearance of responsiveness to beta-adrenergic agonists and the loss of sensitivity to alpha-agonists. These findings are in agreement with previous suggestions that beta-adrenergic receptor activity is modulated by thyroid hormone levels and demonstrate that the specificity of adrenergic modulations of erythropoiesis in culture may accurately reflect the thyroid status of the intact animal.


Alpha-1-antitrypsin deficiency and hepatocellular carcinoma. Forty-two cases of hepatocellular carcinoma (HCC) were examined for the presence of the inclusions of alpha-1-antitrypsin (AAT), which indicate a carrier state for the Pi Z gene. These were found in the non-neoplastic liver tissue of two cases of HCC and in one of the 98 control livers, a difference that is not statistically significant. Typical globules of AAT deficiency were not found in HCC cells. One-quarter of HCCs, however, contained cells which showed diffuse cytoplasmic staining for AAT, a pattern seen also in the non-neoplastic livers.


Viral exposure and abnormal liver function in haemophilia. Several studies have recently documented the presence of persistently abnormal liver function tests in asymptomatic haemophiliacs. While the aetiology is unknown it is possible that repeated exposure to agents transmitted in blood products may be important. This study has attempted to determine the prevalence of viral exposure and its relationship to liver function in this multitransfused group of individuals. The prevalence of viral antibodies with the exception of antibody to hepatitis B surface antigen (anti-HBs) and cytomegalovirus (CMV) was normal when compared to that in the general population. Hepatitis B surface antigen (HBsAg) was not detected, but anti-HBs was found in 83% of patients; 50% of patients had abnormal liver function. However, liver function tests were normal in all patients with mild haemophilia and were only rarely abnormal in patients who had no detectable antibody to CMV, Epstein-Barr virus (EBV), and HBsAg. This study demonstrates that multiple transfusions of blood products, that is, cryoprecipitate and factor concentrates, do not increase the risk of exposure to the viruses studied with the exception of hepatitis B virus.


Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat. Neurophysiological and behavioral measures were obtained from 32 senescent (28--34 mo) and 32 mature adult (10--16 mo) rats. Extracellularly recorded synaptic responses were obtained from electrodes chronically implanted in the fascia dentata and perforant path. The rats were first tested on a circular platform, which favored the use of spatial cues for its solution, and the senescent rats were shown to exhibit poorer memory for the rewarded place. When granule cell synaptic responses were recorded after a single session of very brief high-frequency stimulation, the amount of elevation and time course of decline were equivalent between age groups. Af ter three repetitions, however, the young rats maintained the increased synaptic strength for at least 14 days, whereas the old rats declined after the first session. The amount of synaptic enhancement was statistically correlated with the ability to perform the circular platform task both within and between groups. Furthermore, the aftereffects of the high-frequency stimulation selectively impaired the old rats' spontaneous alternation behavior on a T-maze. Certain other neurophysiological and electroencephalographic measures did not distinguish between age groups. The results are discussed in terms of the synaptic theory of memory formation and of their relevance to the aging process.


Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives. A series of 9-substituted adenine derivatives inhibited adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of a particulate preparation of human blood platelets. A 3--6 fold elevation of adenylate cyclase activity by prostaglandin E1 (PGE1) was inhibited in a concentration-related manner by 9-(tetrahydro-5-methyl-2-furyl) adenine (SQ 22,538), 9-(tetrahydro-2-furyl) adenine (SQ 22,536), 9-cyclopentyladenine (SQ 22,534), 9-furfuryladenine (sQ 4647) and 9-benzyladenine (SQ 218611). The I50 values ranged from 21 microM for SQ 22,538 to 140 microM for SQ 21,611. These same adenine derivatives reversed the inhibition by PGE1 of ADP-induced aggregation and the PGE1-stimulated elevation of adenosine 3':5'-monophosphate (cyclic AMP). The reversal of platelet aggregation inhibition by SQ 22,536 and SQ 4647 was concentration-related with I50 values of 30 microM in each case, whereas SQ 22,534 and SQ 21,611 reversed inhibition by 30% at 100 microM. SQ 22,536, SQ 22,534 and SQ 21,611 also blocked the increase in cyclic AMP levels in a concentration-related manner with I50 values of 1, 4 and 60 microM, respectively. SQ 4647 inhibited the elevation of cyclic AMP by more than 85% at 1000 microM. The adenine derivatives had no effect on platelet aggregation or on cyclic AMP levels in the absence of PGE1. These results provide additional evidence that the inhibition of platelet aggregation by PGE1 is mediated by cyclic AMP.


The stability constants of some metal ion complexes of 3',5'-cyclic AMP. The stability constants of complexes of 3', 5'-cyclic AMP with Mg2+, Ca2+, Mn2+, Ni2+ and Co2+ were estimated at 30 degrees C in solutions of ionic strength about 0.15 containing about 130 mM K+ or tetramethylammonium ions. Values between 13 and 22 M-1 were obtained, indicating that only about 2% of cyclic AMP is present as metal complexes in vivo, but that at commonly used in vitro concentrations of 10 mM bivalent metal ions, 10--20% of cyclic AMP is present as metal complexes. The possible significance of these metal complexes, for example as competitive inhibitors, is discussed.


1,N6-Etheno-2-aza-adenosine 3', 5'-cyclic phosphate: human erythrocyte membrane binding and activation of membrane protein kinase. The relative efficiency of 1,N6-etheno-2aza-adenosine 3', 5'-monophosphate (cyclic 2-aza-epsilon AMP), 1,N6-etenoadenosine 3', 5'-monophosphate (cyclic epsilon AMP) and cyclic AMP in activation of membrane protein kinase and binding to membrane was examined using isolated membranes from human erythrocytes. Cyclic 2-aza-epsilon AMP was 81% as active as cyclic AMP in erythrocyte membrane binding and activation of membrane protein kinase. On the other hand, cyclic epsilon AMP was 37% as active toward membrane protein kinase and 29% toward membrane cyclic AMP binding. Since we have previously shown that the fluorescence of cyclic 2-aza-epsilon AMP is highly sensitive to the polarity of solvents, the high efficiency of cyclic 2-aza-epsilon AMP to substitute for cyclic amp suggests that it may be a suitable microenvironmental fluorescent probe for cyclic AMP binding sites.


Catecholamine-induced desensitization involves an uncoupling of beta-adrenergic receptors and adenylate cyclase. Incubation of 1321N1 astrocytoma cells for 15 min with 1 microM (-)-isoproterenol resulted in a 50-65% loss of isoproterenol-stimulated adenylate cyclase activity. No decrease occurred in basal adenylate cyclase activity or in the density of beta-adrenergic receptors as assessed by (125I)-hydroxybenzylpindolol binding. Concentration-effect studies indicated that the apparent affinity of isoproterenol for inhibition of (125I)-hydroxbenzylpindolol binding was decreased by approximately 10-fold in membranes prepared from cells that had been exposed to 1 umM isoproterenol for 15 min. In the presence of GTP there was a shift to the right of the concentration-effect curve for isoproterenol in control membranes. GTP had little effect on the apparent affinity of isoproterenol in desensitized membranes. In desensitized cells that were subsequently washed free of catecholamine, the decrement in isoproterenol-stimulated adenylate cyclase activity and the decrease in the capacity of isoproterenol to inhibit 125IHYP binding returned to control levels within 15 min. These data are consistent with the hypothesis that an early event in the process of desensitization in 1321N1 cells involves a reversible uncoupling of beta-adrenergic receptors and adenylate cyclase.


The nature of covalent complexes of phosphoproteins with collagen in the bovine dentin matrix. The bovine dentin matrix still contains some noncollagenous proteins after thorough extraction and decalcification. These have been obtained following digestion of the matrix by cyanogen bromide. Peptides containing non-collagenous portions were isolated by chromatography on diethylaminoethyl cellulose columns and fractionated on hydroxyapatite columns. Several fractions were obtained. The principal component was a complex between a highly-phosphorylated serine-aspartic acid-rich protein and a collagen peptide. These collagenous and non-collagenous moieties could not be separated from each other even under highly dissociative solvent conditions. After digestion with collagenase, the resulting phosphoprotein fraction still contained a few residues of hydroxyproline and hydroxylysine, and an enhanced content of proline, compared to the equivalent directly extractable phosphophoryn of the matrix. These data were interpreted as indicating that the phosphophoryn which is not extractable in 0.5M ethylenediaminetetraacetic acid is in fact covalently bound to some specific section of the matrix collagen. The covalent modification of the collagen matrix with highly acidic phosphoproteins may have an important role in the mineralization process.


Effect of donor age on the binding and degradation of low density lipoproteins by cultured human arterial smooth muscle cells. The ability of human arterial smooth muscle cells growing in tissue culture to bind, incorporate and degrade low density lipoproteins (LDL) was studied as a function of cell donor age from 5 to 71 years. LDL binding to cell surface receptors did not change with donor age. However, LDL degradation was significantly decreased in cells from older donors. These results may be relevant to the enhanced accumulation of cellular cholesterol and LDL observed in the aging human artery. Thus, impaired LDL degradation by arterial smooth muscle cells may be one of several factors involved in the accelerated development of atherosclerosis with aging.


Effect of experimental prolongation of life span on lipofuscin content and lysosomal enzyme activity in the brain of the housefly, Musca domestica. The effect of experimental prolongation of life span on the rate of lipofuscin accumulation in the giant neurons of the brain and lysosomal enzyme activity in the head was studied in the male housefly, Musca domestica. The median and the maximum life span of the flies was extended twofold by varying their flight activity. The rate of lipofuscin accumulation was significantly faster in short-lived, high-activity flies as compared to the long-lived, low-activity flies. The maximal content of lipofuscin in the neurons was similar in both groups but was reached at a much older age in the low-activity flies. Age-associated profiles of the activities of the lysosomal enzymes beta-glycerophosphatase and beta-acetylglucosaminidase did not correspond to the mortality characteristics of the housefly populations. The results support the view that lipofuscin represents an integral aspect of aging and may be used as a cellular marker for physiological age whereas lysosomal enzyme activities do not seem to be directly related to the rate of aging.


Sleep in young and old rats.

Sleep in young and old rats. Five young rats, age 152--175 days, and six old rats, age 782--801 days, all of the F-344 strain, were compared by electronic methods for amplitude of slow wave activity during sleep and for other sleep parameters. Unlike humans, who show a pronounced loss of slow wave activity with advanced age, no significant difference in delta activity could be detected between young and old rats. Several hypotheses about the species difference were reviewed. Young and old rats, however, did show several differences in other sleep parameters which parallel those observed in humans. These age-related changes were a moderate decrease in the percent of total sleep time spent in paradoxical sleep, a decrease in the length of sleep bouts, an increase in the number of sleep bouts, and a decrease in the amplitude of the diurnal rhythm of sleep.


Influence of different routes of anti-tumor immunization: alternative induction of tumor immunity and tumor enhancement. Chickens and quails were immunized in parallel either i.v. or intramuscularly (i.m.) with lectin column-purified antigens from chick embryo cells that were transformed in vitro by avain sarcoma virus (ASV). After five to six injections, immunity of the animals was tested by challenge with ASV into the wing webs. Whereas tumor growth was inhibited after i.v. immunization with respect to incidence rate and time of tumor appearance, tumor growth was enhanced after i.m. injection. Animals that were injected with normal cell antigens served as controls. Spleen cells from only those animals that were immunized i.v. exerted a cytotoxic effect in vitro against ASV-transformed cells, whereas spleen cells from i.m. injected animals, in contrast, suppressed such cytotoxicity. The search for serum blocking or arming factors suggested that sera from i.m. injected animals block cellular cytotoxicity whereas sera from i.v. immunized animals render normal spleen cells cytotoxic (arming effect). The use of viruses from different subgroups and of antigens from gp85-lacking ASV-transformed cells indicates that immune effects were obtained against tumor cell surface antigens that differ from the antigen that is involved in virus neutralization (s-gp85).


Phenotypic and functional effects of the motheaten gene on murine B and T lymphocytes. Lymphoid cells from C57BL/6 mice homozygous for the me gene exhibit multiple phenotypic and functional abnormalities from early as one week of age. In the B cell population these include a reduction in the frequency of detectable surface Ig+ cells, alterations in the level of expression of surface IgM and IgD, an increase in the frequency of large cells, plasma cells and TNP-specific plaque forming cePS. Together these findings provide strong evidence for polyclonal activation of B cells. The high level of expression of xenotropic MuLV gp70 by me/me spleen and lymph node cells provides further evidence for lymphoid cell activation. In preliminary studies, me/me T cells appeared to be phenotypically and functionally less affected by the me gene. The distribution of Thy 1.2 on the surface of spleen and lymph node T cells varied from low to normal and the mitogenic responses to Con A and PHA were depressed. It remains to be determined what the basic deficiency in me/me mice is and whether it affects primarily B cells or all lymphoid cells.


Antigenic properties and molecular weights of murine leukemia virus-binding proteins. A murine T lymphoma cell line, WEHI-22, has been studied for the presence of murine leukemia virus-binding proteins and for the presence of cell surface molecules that share antigens with mouse immunoglobulins. With surface radioiodination, detergent disruption, and immunoprecipitation, a 60 to 70,000-dalton molecule has been described that is recognized by chicken anti-mouse immunoglobulin serum. In competition experiments this molecule cross-reacts with highly purified mouse IgM myeloma proteins. A cell surface molecule of similar size can be shown to bind to mouse leukemia viruses. Pre-precipitation of the WEHI-22 cell surface material with chicken anti-mouse immunoglobulin removes the material binding to leukemia viruses.


Hybrid resistance to BALB/c plasmacytomas. II. Radiation sensitivity and silica insensitivity of resistance to MPC-11. Resistance to the BALB/c plasmacytoma MPC-11 by F1 hybrids between BALB/c and four C57BL/10 congenic resistant strains was abrogated or reduced by 450 rads total body irradiation but was unaffected by intravenous injection of 3 to 4 mg of silica. The results are consistent with the idea that hybrid resistance to MPC-11 depends on an active immune response and is different from Hh-1 controlled hybrid resistance.


Lymphocyte activation by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA, a highly active tumor-promoting agent, is an effective mitogen for primate peripheral blood lymphocytes. Optimal stimulation of human lymphocytes was obtained 4 days after the addition of TPA at a concentration of 7.5 ng/ml. Lymphocyte fractionation experiments demonstrated that both T and B cells incorporated 3H-thymidine significantly in response to TPA. Lymphocyte blastogenesis was not due to the reactivation of latent herpesviruses by the tumor promoter, since similar responses to TPA were obtained with virus-genome positive or negative cells. Increased levels of DNA synthesis were observed when TPA was added to marmoset, baboon, rhesus monkey, or chimpanzee peripheral blood lymphocytes. Canine peripheral blood lymphocytes and spleen cells from guinea pigs, rats, and mice were not stimulated by TPA. These observations suggest that TPA-induced lymphocyte blastogenesis may be useful for studies of lymphocyte activation and of the molecular mechanisms of action of tumor-promoting phorbol esters.


Mechanisms of leukemogenesis. I. Generation of autoreactive lymphocytes in response to a murine leukemia virus. Examined in this paper is the capacity of 334C murine leukemia virus (MuLV) to stimulate the generation of virus-specific cytotoxic effector cells in mice of the C57BL/6 strain that are relatively resistant to Friend, Moloney, and Rauscher (FMR) MuLV-induced leukemia, and in BALB/c mice that are relatively susceptible to leukemia induced by FMR MuLV. Generation of cytotoxicity requires in vivo administration of the virus followed by in vitro culture of lymphoid cells from virus-injected animals. Lymphoid cells from MuLV-resistant C57BL/6 donors develop high levels of specific cytotoxicity after secondary in vitro stimulation with syngeneic MuLV-induced tumor cells. Cells derived from these same donors, cultured in the absence of MuLV-induced tumor cells, fail to exhibit cytotoxicity. Secondary in vitro stimulation of lymphocytes from MuLV-susceptible BALB/c animals results not only in generation of cytotoxic reactivity against syngeneic MuLV-induced tumor cells but also induces apparently autoreactive effector cells capable of lysing other H-2d tumor cells as well as normal peritoneal cells bearing H-2d antigens. Moreover, generation of cytotoxicity by BALB/c lymphocytes occurs whether or not MuLV-induced tumor cells are included in the secondary culture system.


Hypocomplementemia associated with naturally occurring lymphosarcoma in pet cats. Eighty cats were classified by indirect immunofluorescence and histologic diagnosis into four categories: normal, feline leukemia virus (FeLV) infected; normal noninfected; lymphosarcoma-FeLV infected; lymphosarcoma, no FeLV present. All viremic cats with lymphosarcoma were found to be hypocomplementemic and activation of the complement system had occurred via the classical pathway. Sera of cats with lymphosarcoma in the absence of FeLV had varying levels of total hemolytic complement (TCH50) ranging from normal to hypocomplementemic. Approximately 50% of the cats that were viremic but histologically and clinically free of disease had TCH50 levels within normal range, and the remainder exhibited varying degrees of hypocomplementemia.


Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide. The adjuvant effects of mycobacteria can be replaced by more chemically defined isolates of the cell walls including a water soluble fraction (WSA) and by the synthetic analog N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal structure required for adjuvanticity. These compounds can directly activate macrophages as determined by an increase in spreading and adherence and by an elevated synthesis of the enzyme collagenase. Moreover, this increase in collagenase production is modulated by enhanced production of prostaglandins that influences intracellular levels of cyclic AMP. In addition, both MDP and WSA induced macrophages to produce a biologically active mediator that triggers quiescent fibroblasts into active proliferation. It thus appears that a mechanism for mycobacterial adjuvant action as determined with MDP and WSA is via activation of macrophages, which may then precipitate a multiplicity of other reactions resulting in enhanced immune phenomena. Furthermore, the granulomatous and fibrotic reactions associated with mycobacterial infection may be a consequence of this direct activation of macrophages.


Antibody blockade of lysis by T lymphocyte effectors generated against syngeneic SV40 transformed cells. Antibody blockade of cell-mediated lysis was used to probe the cell surface structures recognized by T lymphocytes generated to syngeneic SV40 virus-transformed mouse cells. Alloantisera to H-2 antigens were highly effective in inhibiting lysis. Anti-H2 antibody blockade of lysis was haplotype specific even on transformed F1 target cells. Inhibition occurred only when antibody was bound to the target cell. Antibody binding to the effector did not inhibit lysis. Inhibition required that antibody be bound to the H-2 molecule itself; antibody to molecules associated with H-2, such as beta2-microglobulin, had no effect. Attempts to block lysis by using antisera to known virus-coded molecules were uniformly unsuccessful. These results are discussed in light of current controversy concerning the nature of the SV40 virus-specific transplantation antigen.


Variations in expression of xenotropic murine leukemia virus genomes in lymphoid tissues of NZB mice. Lymphocytes from Thy, Sp, LN, and BM and NZB mice were tested for expression of X-MuLV genomes as cell surface gp70 (XenCSA) or infectious virus. The results demonstrate major dissociations for these parameters of X-MuLV expression in different lymphoid compartments and suggest that factors involved in T lymphocyte differentiation modify the levels of expression in these two modes.


Enhanced immune response late in primary cytomegalovirus infection of mice. Murine cytomegalovirus (MCMV) infection has been previously shown to depress humoral and cell-mediated functions to non-MCMV antigens. In this report we show that in C3D2 mice undergoing nonlethal primary infection the depressed anti-sheep RBC plaque-forming cell (PFC) response is followed by an enhanced PFC response. Infected mice often generated twice the number of PFC per spleen than that of control mice. Total numbers of spleen cells as well as the recovery of virus from spleens of infected mice did not distinguish the depressed from the enhanced phase of the response. Investigation of the kinetics of the response revealed a defect in shutdown regulation. This enhanced PFC response during primary MCMV infection was not reflected in measurements of serum hemagglutinin. These findings suggest that MCMV induces an impairment of immunoregulation.


Differentiation antigens on normal and Abelson virus transformed lymphocytes. Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.


The effects of mitogens on the expression of Epstein-Barr virus antigens in human lymphoid cell lines. Treatment of human lymphoblastoid cells with either phytohemagglutinin (PHA), concanavalin A, Staphylococcus protein A, or polyinosinic acid-polycytidylic acid, in combination with 5-iodo-2' deoxyuridine (IUdR) markedly increased the expression of Epstein-Barr virus (EBV) early antigen (EA) relative to IUdR alone. Such treatment did not, however, modify the production of virus capsid antigen in any of the lymphoid cell lines tested. The effect of PHA on EA induction in Raji cells was not accompanied by changes in the incorporation of labeled precursors into cellular DNA, or in the intracellular concentration of either adenosine 3'5' cyclic monophosphate or guanosine 3'5' cyclic monophosphate. However, those mitogens that stimulated EA expression in Raji cells also increased the fluorescence polarization of 1,6 diphenyl 1,3,5-hexatriene-labeled Raji cells. The possible role of cell surface changes in the mitogen activation of latent EBV in human lymphoblastoid cells is discussed.


Fractionation and immunologic assessment of KCl-extracted cardiac antigens in coxsackievirus B3 virus-induced myocarditis. Hypertonic salt extracts prepared from the heart tissues of adolescent CD-1 mice were fractionated on Sephadex G-100 columns. Two separate fractions were obtained. Fraction I, containing the antigenic immunoreactive activity, was able to inhibit the migration of CVB3-PPD immune mouse peritoneal exudate cells (IMPEC) as well as PEC from mice infected with CVB3 virus alone. Fraction II did not have antigenic activity as assessed by the agarose droplet cell migration inhibition assay. As controls, Fraction I prepared from the livers of spleens of CVB3-infected CD-1 mice was unable to inhibit the migration of CVB3 IMPEC. Unimmunized or "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited by Fraction I. Antibodies prepared against Fractions I and II were unable to neutralize CVB3m virus in the plaque reduction test, and polyacrylamide gel analysis revealed multiple bands in 10% SDS gels.


Enzyme immunoassay for feline oncornavirus-associated cell membrane antigen (FOCMA) and detection of FOCMA in cell extract by enzyme immunoassay inhibition test. An enzyme immunoassay (EIA) for FOCMA has been developed. The assay uses alkaline phosphatase-conjugated rabbit anti-cat IgG as the second antibody and p-nitrophenyl phosphate as the substrate for the enzyme to detect cat FOCMA antibody bound to the target cells. In comparison with the indirect immunofluorescence (IIF) test, which was originally used for FOCMA assay, our results showed a good correlation between the two methods. The EIA gives a more objective measure of FOCMA reactivity than does IIF. FOCMA was successfully extracted from FOCMA-positive cell membranes by 0.5% Triton X-100 and further fractionated by ammonium sulfate. The FOCMA activity was assayed by IIF and EIA inhibition test. Most of the FOCMA activity was found in the fractions precipitated by 30% and 50% ammonium sulfate saturation.


Beta adrenergic receptor binding on polymorphonuclear leukocytes in atopic dermatitis. It has been postulated that the patient with atopic dermatitis has defective beta adrenergic receptor function. However, a more generalized defect is suggested by the observation that cyclic AMP generation is diminished in these patients following stimulation with both isoproterenol and PGE1. To determine the nature of this abnormality, we measured beta adrenergic receptor binding directly on polymorphonuclear leukocyte membranes using the radiolabeled beta adrenergic antagonist (-) [3H]dihydroalprenolol (DHA). DHA binding was studied in 6 mild and 9 moderate-to-severe atopic dermatitis patients, and 8 normal controls using a subsaturating concentration of DHA (0.5 nM) to estimate receptor affinity and a saturating concentration of DHA (30 mM) to determine the total number of receptors per cell. No significant differences (p greater than .05) were found in the total number of receptors per PMN between the control population (805 +/- 95) and the mild atopic dermatitis patients (745 +/- 91) or the moderate to severe group (621 +/- 79). In addition, no significant differences in receptor affinity were found among any of the 3 study groups. These findings suggest that beta receptor binding in atopic dermatitis is normal. Reduced cyclic AMP generation in atopic dermatitis PMN leukocytes would appear to be due to a defect distal to the beta adrenergic receptor itself.


Migration of epithelial cells in the small intestine of mice perorally infected with coxsackievirus B5. The rate of cell migration in the small intestine during enteric viral infections has not been assessed previously. CD-1 mice (33 days old) were infected perorally with 1.0 X 10(8) plague-forming units of coxsackievirus B5 and 12 hr later were injected intraperitoneally with 2 micron Ci of [3H]thymidine/g of body weight. After 2, 12, 24, 48, 60, and 72 hr, mice were killed, and the small intestine was removed. Specimens obtained at each interval were examined by radioautography; similar specimens were titrated for virus by plaque assay in HeLa cells. In mice perorally infected with coxsackievirus B5, epithelial cells migrated from crypt to villus tip in 60 hr, as compared with 48 hr in uninfected control mice and 24 hr previously reported for mice perorally infected with enteric bacteria (e.g., Salmonella typhimurium). Virus was recovered from intestinal tissue, but no inflammatory response in the limina propria was apparent. These observations are consistent with previous report that substrate absorption rates may be altered during viral and bacterial enteric infection.


Simultaneous acute infections with hepatitis A and hepatitis B viruses in a chimpanzee. The unexpected occurrence of a hepatitis B virus (HBV) infection in a chimpanzee experimentally inoculated with hepatitis A virus (HAV) provided an opportunity to examine the course of simultaneous acute infections with both agents. A chimpanzee inoculated intravenously with HAV developed elevated levels of aminotransferases in serum, detectable excretion of hepatitis A antigen in feces, and a marked antibody response to HAV. During the acute phase of this experimentally induced infection with HAV, the chimpanzee simultaneously developed an HBV infection. The latter was characterized by jaundice, a second increase in levels of aminotransferases in serum, and the appearance in serum of hepatitis B surface antigen (HBsAg), hepatitis B e antigen, antibody to hepatitis B core antigen, and, later, antibody to HBsAg. During the acute phase of both HAV and HBV infections, marked histopathologic inflammatory changes were observed in serial liver biopsy specimens. In this chimpanzee, the concurrent acute infection with both HAV and HBV occurred in association with marked liver damage.


Activation of reticuloendothelial cells following infection with murine cytomegalovirus. Macrophage and reticuloendothelial system function was investigated in adult, female C3H mice during the course of a nonlethal infection with murine cytomegalovirus (MCMV). Peritoneal macrophages harvested from mice infected three to 21 days previously were tumoricidal in vitro for a syngeneic mammary tumor cell line and showed antiviral activity by suppressing replication of vaccinia virus in a fibroblast monolayer. In addition, macrophages harvested from animals on days 7 and 14 after MCMV infection manifested augmented phagocytosis of yeast particles in vitro. Furthermore, enhanced levels of chemiluminescence were emitted during phagocytosis of zymosan particles by macrophages harvested on days 3 and 7 after MCMV infection. Finally, enhanced protection against Listeria monocytogenes was demonstrated in mice on days 7, 14, and 21 after MCMV infection. These findings support the concept of activation of the reticuloendothelial system during viral infection.


[Studies on the refractory coating materials for preventing scale formation (author's transl)]. In casting of high-fusing alloys such as cobalt-chrome and nickle-chrome alloys, the reaction between the investing mold and high-fusing molten alloys suffers the disadvantage of the scale formation. The various coating materials are used to prevent the scale formation. The experiments have been carried out to prove the effectiveness of seven commercialized coating materials. And the reaction between the coating materials and high-fusing molten alloys was analyzed by X-ray diffractometer. Results were as follows: 1) The refractory compositions of seven commercialized coating materials were composed of alpha-quartz (SiO2), zircon (ZrSiO4) and zirconium oxide (ZrO2). 2) Dainainvest (Ohara) mainly containing zircon indicated the reduction in surface roughness due to coating. Although zircon was slightly observed on the cast surface, the product of reaction was not detected. 3) Liquid investment (Nobilium) and Paint investment (Shofu) having about equal amounts of alpha-quartz and zircon showed as reduction in surface roughness as Dainainvest. The product of reaction was not observed and the cast surface was smooth. 4) Quick request (Shiken), Pre-coat (Niranium) and Ethymay B (Kamemizu) composed of alpha-quartz had not appreciably effectiveness. The cast surface was rough due to production of picrochromite (MgCr2O4) and eskolaite (Cr2O3)in the reaction between molten alloys and coating materials. 5) Hi-coat with zirconium oxide did not point out the advantage in spite of high refractory composition and showed little reduction in surface roughness due to coating.


[Preparation of hard crown and bridge resin without PMMA powder (author's transl)]. Elimination of PMMA powder to prepare better hard crown and bridge resin had been pointed out by the authors even though polyfunctional methacrylates were used. Application of hydrophobic coloidal silica instead of PMMA pearl with triethyleneglycol dimethacrylate and Bis-GMA was studied. Polyermization was carried out in the hot air stream at 120 degrees approximately 130 degrees C or in a pressure vessel at 4.5 kg/cm2 gauge, 100 degrees C. Mechanical properties, brinell hardness, compressive strength, an amount of water sorption, comparative average abrasion loss depth with those of PMMA, were measured. They were improved very much by this new system.


Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats. Isolated rat livers were perfused for four hours in a recirculating system containing washed rat erythrocytes. Biologically screened radioiodinated rat high density lipoproteins (1.090 < d < 1.21 g/ml) were added to the perfusate with different amounts of whole serum to supply unlabeled rat high density lipoproteins. The protein moiety of the lipoprotein contained more than 95% of the radioiodine. The fraction of apolipoprotein mass degraded during the perfusion was quantified by the linear increment of non-protein-bound radioiodine in the perfusate, corrected for the increment observed during recirculation of the perfusate in the absence of a liver. The small amount of (131)I secreted into bile was added to calculate the fractional catabolic rate. The fractional catabolic rate ranged from 0.22 to 0.63% per hour in 12 experiments and was inversely related to the size of the perfusate pool of high density apolipoprotein. The absolute catabolic rate of high density apolipoprotein (fractional catabolic rate x pool size) in three livers in which the concentration of rat HDL in the perfusate approximated that in intact rats was 69.5 +/- 10.4 micro g hr(-1) (mean +/- SD). The rate of disappearance of cholesteryl esters of rat high density lipoproteins (labeled biologically by injecting donor rats with [5-(3)H]mevalonic acid) from the liver perfusate did not exceed that of the apoprotein component. These rates were compared with catabolic rates for rat high density lipoproteins in intact rats. Fractional catabolic rate in vivo, obtained by multicompartmental analysis of the disappearance curve of (131)I-high density apolipoprotein from blood plasma, was 11.9 +/- 1.3% hr(-1) (mean +/- SD). Total catabolic rate in vivo (fractional catabolic rate x intravascular pool of high density apolipoprotein) was 986 +/- 145 micro g hr(-1) (mean +/- SD). The results suggest that only a small fraction of high density lipoproteins in blood plasma of rats is degraded directly by the liver.-Sigurdsson, G., S-P. Noel, and R. J. Havel. Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats.


Translation in vivo and in vitro of proteins resembling apoproteins of rat plasma very low density lipoprotein. Antibodies raised against rat plasma apoVLDL and a purified fraction of arginine-rich peptides (ARP) were labeled with Na125I and were shown to bind to polyribosomes isolated from rat liver. Antibody fractions enriched by selective affinity chromatography exhibited increased levels of binding to polysomes. Anti-apoVLDL immunoreactivity was further resolved into anti-ARP and anti apoB components, each reactive with a distinct polysome population. Binding was specific for rat polysomes, and was directed toward nascent polypeptide chains. About 2% of normal rat liver polysomes were recovered by indirect immunoprecipitation with anti-apoVLDL. Ribonucleic acid (RNA) extracted from this immunoprecipitate contained species with polyadenylate (poly[A] sequences characteristic of eukaryotic messenger RNA (mRNA). These species, purified by affinity chromatography on poly(U)-Sepharose, stimulated the in vitro synthesis of immunoprecipitable apoVLDL-like proteins by about 17-fold when compared to unfractionated rat liver mRNA. Most of the in vitro translation products precipitated by purified anti-ARP migrated identically on polyacrylamide gel electrophoresis with unlabeled purified ARP. Some implications of these findings with respect to plasma VLDL biosynthesis are discussed.


Characterization of a model of dietary-induced hypertriglyceridemia in young, nonobese rats. Healthy, nonobese, young rats developed hypertriglyceridemia (mean triglyceride levels of 250 mg/dl) following consumption of a sucrose-lard diet. The hypertriglyceridemia was apparent three days after start of the diet and persisted throughout the 4-week experimental period. Body weight, liver weight, and serum glucose levels were similar in animals eating either the sucrose-lard diet or standard rat chow. On the other hand, serum free fatty acid levels were slightly increased and serum insulin levels were substantially increased in animals eating the sucrose-lard diet. Determination of very low density lipoprotein turnover revealed that total triglyceride secretion in rats eating the sucrose-lard diet was significantly (P < 0.01) increased over that of rats eating standard chow. Direct measurement of hepatic and intestinal very low density lipoprotein secretion indicated that the observed rise in total triglyceride secretion was secondary to increased secretion of very low density lipoproteins by the liver. Finally, lipoprotein lipase activity of adipose tissue from rats eating the sucrose-lard diet was equal to, or greater than (depending upon sampling time), the activity of the enzyme from adipose tissue of rats eating the control diet. These data indicate that young, nonobese, rats develop hypertriglyceridemia when they ingest a sucrose-lard diet, and that the rise in plasma triglyceride levels results from an increase in hepatic very low density lipoprotein secretion. The dietary-induced hypertriglyceridemia is associated with elevated serum insulin levels, and, as such, may provide a useful animal model to use in studies aimed at defining the pathogenesis of endogenous hypertriglyceridemia in man.-Reaven, G. M., T. R. Risser, Y-D. I. Chen, and E. P. Reaven. Characterization of a model of dietary-induced hypertriglyceridemia in young, nonobese rats.


Regulation of plasma lecithin:cholesterol acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal. Plasma lecithin:cholesterol acyltransferase (LCAT) activity is increased during the clearance phase of alimentary lipemia induced by a high-fat test meal in normal subjects. Ultracentrifugal fractionation of high density lipoproteins (HDL) into HDL(2), HDL(3), and very high density (VHD) subfractions followed by analyses of lipid and protein components has been accomplished at intervals during alimentary lipemia to seek associations with enzyme changes. HDL(2) lipids and protein increased substantially, characterized primarily by enrichment with lecithin. HDL(3), which contain the main LCAT substrates, revealed increased triglycerides and generally reduced cholesteryl esters which were reciprocally correlated, demonstrating a phenomenon previously observed in vitro by others. Both changes correlated with LCAT activation, but partial correlation analysis indicated that ester content is primarily related to triglycerides rather than LCAT activity. The VHD cholesteryl esters and lysolecithin were also reduced. Plasma incubation experiments with inactivated LCAT showed that alimentary lipemic very low density lipoproteins (VLDL) could reduce levels of cholesteryl esters in HDL by a nonenzymatic mechanism. In vitro substitution of lipemic VLDL for postabsorptive VLDL resulted in enhanced reduction of cholesteryl esters in HDL(3) and VDH, but not in HDL(2), during incubation. Nevertheless, augmentation of LCAT activity did not result, indicating that cholesteryl ester removal from substrate lipoproteins is an unlikely explanation for activation. Since VHD and HDL(3), which contain the most active LCAT substrates, were also most clearly involved in transfers of esters to VLDL and low density lipoproteins, the suggestion that LCAT product lipoproteins are preferentially involved in nonenzymatic transfer and exchange is made. The main determinant of ester transfer, however, appears to be the level of VLDL, both in vitro and in vivo. Rose, H. G., and J. Juliano. Regulation of plasma lecithin: cholesteryl acyltransferase in man. III. Role of high density lipoprotein cholesteryl esters in the activating effect of a high-fat test meal.


A comparison of delipidated sera used in studies of sterol synthesis by human mononuclear leukocytes. Sterol synthesis in human mononuclear leukocytes is stimulated by delipidated serum. Synthesis in media containing serum delipidated by three different methods is compared. Significant differences between subjects are shown and the differences are maximized by measuring synthesis in serum delipidated by extraction with butanol-diisopropyl ether 40:60 and diethyl ether.


The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes. III. B-cell dependence for T-cell proliferation. The nature of the helper lymphocytes in lymphocytosis-promoting factor (LPF)-induced proliferation was explored. Removal of macrophages from adherent splenocytes by either carbonyl-iron incubation or passage through Sephadex G-10 columns did not affect their synergistic function. Nor did cytolysis with Thy-1.2 antiserum and complement. The helper cells were found to be surface immunoglobulin-positive (sIg+) because they are retained by anti-Ig columns, susceptible to lysis by rabbit anti-mouse immunoglobulin and complement, and occurred in the sIg+ fractions of splenocytes after separation on the fluorescence-activated cell sorter. Further delineation of the surface markers on helper cells showed that complement receptors are not the determining marker for synergistic function. The requirement for B-helper cells in the stimulation of T lymphocytes by LPF is unique for a mouse of T-cell mitogen.


Association of circulating retroviral gp70-anti-gp70 immune complexes with murine systemic lupus erythematosus. Endogenous retroviral gp70 was investigated as a participant in the pathogenesis of a lupus-like disease that spontaneously develops in four kinds of mice (NZB, NZB x W MRL/1, and male BXSB). Sera from these strains contain a heavy form of gp 70 that varies in sedimentation rates from 9S to 19S in sucrose density gradient analysis and appears with the onset of disease and persists throughout its course. Immunologically normal strains of mice do not develop rapidly sedimenting gp70 by 8-10 mo of life. The fact that the heavy gp70 is selectively absorbed with anti-IgG antibodies or with Staphylococcus aureus protein A suggests that it is complexed with antibodies. The incidence and quantities of these gp70 ICs rise with the progression of disease in all strains with lupus. These findings suggest that Ig-complexed heavy gp70 may be involved in the pathogenesis of glomerulonephritis of mice with SLE.


Plasma levels of immunoreactive parathyroid hormone in dogs chronically exposed to low levels of cadmium chloride. A previous report had suggested that chronic cadmium intoxication interfered with calcium accumulation in bone and suggested that the mechanism may be a parathyroid-induced bone resorption. We measured C-terminal parathyroid hormone levels in the blood of four male standardized research beagles chronically exposed during 6 months to 25 ppm cadmium chloride in their drinking water while at the same time we performed measurements of the parameters of haversian bone remodelling activity. We failed to demonstrate any statistically significant difference in levels of this hormone in the experimental dogs before or during the exposure, or between samples obtained from the experimental dogs at the end of their period of exposure and normal untreated control dogs from the same colony. We conclude that in the early stages of chronic cadmium intoxication before there is evidence suggestive of intestinal or renal malfunction that may secondarily involve the parathyroid glands, the alteration observed in haversian bone remodelling rates is not mediated through abnormalities of function of the parathyroid glands.


Mechanism of rejection of virus persistently infected tumor cells by athymic nude mice. Cell lines known to be tumorigenic in the nude mouse were modified by rendering them persistently infected (P.I.) with a variety of RNA viruses, including measles, mumps, vesicular stomatitis virus, and influenza. Although as few as 100 HeLa or BHK cells produced tumors in 100% of nude mice, as many as 2 x 10(7) of the same cells P.I. with viruses failed to produce tumors. An active host response responsible for restricting the growth of the P.I. cells was suggested by the findings of marked mononuclear cell infiltrates at the inoculation sites and the inability of irradiated nude mice to reject them. An analysis of the in vitro cytotoxic activity of spleen cells from normal nude mice indicated that: (a) P.I. cell lines, but not uninfected cell lines, were susceptible to spontaneous cytotoxicity; (b) in vivo inoculation of P.I. lines induced an enhanced cytotoxic activity for P.I. targets in vitro, and this induction was not specific either for inducing virus or cell line; and (c) the effector cell had the characteristics for natural killer (NK) cells. Although the specificity of recognition of the various P.I. cell lines remains unclear, cold competition experiments indicated that blocking the killing of one P.I. cell line, e.g. HeLa-measles, could be achieved only by unlabeled homologous cells, i.e. HeLa-measles, and not by uninfected cells or other P.I. lines. A variant subline of BHK cells P.I. with VSV was selected for its ability to withstand the rejection process in nude mice. These cells formed metastatic and invasive tumors in nude mice. Although they were the most potent inducers in vivo of NK cell activity against various P.I. targets, they were the most resistant of the P.I. lines to NK cell cytotoxicity in vitro. In this system there was a good correlation between tumor rejection in vivo and susceptibility to NK cells in vitro. The present results suggest that NK cells may play a significant role in both rejection of tumor cells, and in resistance to viruses, particularly persistent infections.


Expression of xenotropic murine leukemia viruses as cell-surface gp70 in genetic crosses between strains DBA/2 and C57BL/6. Flow microfluorometry was used to assess levels of xenotropic murine leukemia virus envelope-related cell-surface antigens (XenCSA) expressed on lymphocytes of mice derived from crosses between C57BL/6 (B6) and DBA/2 (D2); 24 recombinant inbred strains (BXD RIs) and 62 backcross mice were studied. The results suggest that XenCSA expression is affected by more than one gene but that the predominant influence is exerted by a single semidominant gene apparently located on chromosome 4 at or in close proximity to the Fv-1 locus. Studies of spontaneous virus production in B6D2F1 X D2 mice suggest that this locus may also affect production by spleen cells of xenotropic MuLV registering in a fluorescent antibody assay of mink lung cells.


The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes. IV. Generation, characterization, and specificity of cytotoxic lymphocytes. Cytotoxic effector lymphocytes were induced in cultures of mouse spleen or lymph node cells by lymphocytosis promoting factor (LPF). The LPF-activated cytotoxic cells: (a) were not generated unless proliferation occurred; (b) sedimented in the lighter density fraction of a bovine serum albumin gradient; (c) were large, blast-like cells; and (d) were lysed by Thy-1.2 antiserum plus complement and, therefore, were T cells. Neither LPF alone nor supernates from stimulated cultures were cytotoxic. Unlike the situation with concanavalin A and phytohemagglutinin P, LPF-stimulated cytotoxic effector lymphocytes required no further addition of mitogen for maximal cytotoxicity. The effector cells displayed specificity, destroying only allogeneic but not syngeneic normal cells; in the case of tumor cells, both allogeneic and syngeneic cells werelysed in the absence of added mitogen. The reason for differentiated cytotoxicity toward syngeneic tumor and normal cells is not clear but may have some relevance to in vivo tumor rejection initiated by Bordetella pertussis.


Tumors induced by murine sarcoma virus contain precursor cells capable of generating tumor-specific cytolytic T lymphocytes. Leukocyte fractions extracted from the tumor mass and the lymphoid organs of C57BL/6 (B6) mice carrying murine sarcoma virus-induced tumors contained primed cytolytic T-lymphocyte (CTL) precursor cells, in addition to active cytotoxic T cells. These leukocyte fractions gave a secondary response when stimulated in vitro with syngeneic tumor cells, generating large numbers of specific CTL. The activity of these CTL (H-2b) was apparently H-2-restricted, because it was ineffective on tumor targets bearing strongly cross-reacting tumor-specific antigens but with the H-2d haplotype. Furthermore, only H-2b cells bearing the Friend, Moloney, Rauscher-associated antigen, such as Rauscher leukemia virus-induced RBL-5 cells and Friend leukemia virus-induced HFL/b cells, were lysed efficiently. B male GV cells (H-2b cells induced by Gross leukemia virus) were not affected by the same CTL. We propose the existence of a dynamic state involving the migration of primed CTL precursor cells between the lymphoid organs and the tumor mass, as well as the differentiation of these precursor cells within the tumor mass into highly specific CTL.


Systemic lupus erythematosus clinically resembling multiple sclerosis and with unusual pathological and ultrastructural features. A case of systemic lupus erythematosus is described which clinically resembled multiple sclerosis and in which the lesions were restricted to the central nervous system. The necropsy findings of vascular thickening and necrosis in the spinal cord and in a posterior nerve root explain the main clinical abnormalities. Clinical signs of the terminal peritonitis secondary to cholecystitis were absent or minimised probably because of the steroid therapy and spinal cord necrosis. Primary demyelination was not demonstrated though electronmicroscopy revealed lattice fibrillar inclusions within a few myelin sheaths. An unusual ultrastructural feature was the finding of "rod-shaped tubular bodies" in large numbers in the endothelial cells of cerebral blood vessels. The incidence and morphology of these organelles are compared with those of the intracisternal tubuloreticular structures (TRS) commonly found in systemic lupus erythematosus.


Nervous system complications of herpes zoster: immunofluorescent demonstration of varicella-zoster antigen in CSF cells. In nine out of 11 patients with herpes zoster and neurological symptoms varicella-zoster antigen was demonstrated in CSF cells by indirect immunofluorescent staining. Cerebrospinal fluid cytology was in most cases highly suggestive of viral infection. This study demonstrates the value of both immunofluorescent staining on CSF cells and CSF cytology, as corroboration of an otherwise exclusively serologically based virological diagnosis.


Intracranial pressure in the normal monkey while awake and asleep. Intracranial pressure (ICP) was recorded continuously by telemetry in seven normal monkeys trained to eat, sleep, and live in a primate chair. Electroencephalography, electromyography, and blood pressure were also measured by conventional means. During wakefulness and all stages of sleep except desynchronized sleep, the ICP record showed small short-term variations in pressure. However, during desynchronized sleep, the mean ICP rose on the average to 170 +/- 6 mm H2O above the ICP levels in the other states of sleep, and the pulsation pressure variation increased by a factor of three. The episodes occurred 10 +/- 2 times during the night and lasted for 6.8 +/- 1.4 minutes, during which the average systemic blood pressure decreased by 19 +/- 1.6 mm Hg. These ICP waves occurring during desynchronized sleep resemble the plateau waves described by Lundberg, but are of smaller magnitude and they appear to be a normal characteristic of sleep in the macaque monkey. Bilateral sympathectomy of the superior cervical ganglia in four of the monkeys did not alter significantly the duration, amplitude, or frequency of occurrence of the ICP waves during desynchronized sleep.


Spinal subarchnoid metastatic spread from non-neuraxial primary neoplasms. Case report. A rare case of metastasis to the spinal subarachnoid space from a non-neuraxial primary tumor is presented. Dissemination was shown by computerized tomography to be via the cerebrospinal fluid from secondary deposits in the central nervous system and meninges. This route would seem to be the most common mode of spread to the spinal subarachnoid space.


Effect of dietary supplementation of iron and ascorbic acid on lead toxicity in rats. In our laboratory, the protective and therapeutic effects of surplus dietary iron and ascorbic acid on cadmium toxicity in rats have been studied and in this experiment, an effect of surplus iron and ascorbic acid on lead toxicity was examined. In young rats ingesting a diet containing 500 ppm of lead, growth retardation and anemia were observed. Suplementation of 400 ppm of iron and 1% of ascorbic acid to the lead containing diet prevented the growth depression and anemia and caused reductions of concentrations of lead in the kidney and tibia. Whereas, addition of 50 ppm of cadmium to the lead containing diet aggravated the growth retardation and anemia, but reduced the concentrations of lead in the kidney and tibia. Dietary supplementation of iron to the lead containing diet prevented the growth depression and anemia and reduced the accumulation of lead in the kidney, however the supplementation of ascorbic acid alone did not show any ameliolative effects. Rats were fed the lead containing diet and then transferred to the basal diet with or without iron and ascorbic acid. Recoveries from the growth retardation and anemia were not observed in rats within a week after the transfer to the non-lead diet with or without iron and ascorbic acid. These results suggest that iron prevents the growth depression and anemia in rats ingesting lead by an inhibition of lead asborption.


Effects of a protein-free diet on the changes in cyclic AMP and cyclic GMP levels induced by immunization in splenic T and B lymphocytes in the rat. The levels of cyclic nucleotides, cAMP and cGMP, were determined in splenic lymphocytes of normally fed (N) and protein deprived (PD) rats before and at different time intervals after a single injection of sheep erythrocytes. Assays were performed with protein binding methods on unseparated as well as on T and B cells fractionated by filtration through nylon wool. The cAMP levels increased in unfractionated cells and in T and B lymphocytes 2 hours following immunization of N rats. Another rise in cAMP levels occurred after 3 days in B lymphocytes, but there was also a simultaneous increase of the cGMP levels in preparations of unfractionated cells and in B lymphocytes. The PD diet suppressed or delayed most of the aforementioned changes. Thus, the immunodepressive effect of such a diet may be ascribed to the inhibition of both the early signal (increase of cAMP levels and of cAMP/cGMP ratio) leading to T and B differentiation and the later signal (increase of cGMP levels) which initiates antibody production.


Malignant fibrous histiocytoma of the maxillary sinus. A rare localization of a malignant fibrous histiocytoma is presented. Review of the literature showed that the biological behavior of this group of tumors is obscure. The histomorphologic pattern does not always reflect the malignant character, the tendency for recurrence, and the metastasizing potential of the individual tumor. Radiotherapy is ineffective, so the surgeon must opt for radical excision. Additional clinical reports, long-term follow-up, and electron microscopic studies are needed to establish the biological behavior of this group of tumors.


Normal growth despite abnormalities of growth hormone secretion in children treated for acute leukemia. We have studied the relationship between abnormalities of the growth hormone-somatomedin axis and growth in  26 children previously treated for acute lymphatic leukemia. Each child had previously received cranial irradiation, was in complete clinical and hematologic remission, and off all drugs. The mean standing height SDS of the 26 children was significantly less than normal. There was no significant difference between the mean standing height SDS, height velocity SDS, somatomedin activities, and degree of bone age retardation between the 17 children who received the higher dose of cranial irradiation (Group 1) and the nine who had the lower dose of cranial irradiation (Group II). Furthermore, there was no significant reduction in mean height velocity SDS, somatomedin activity, or bone age in either group when compared to normal age-matched controls. The peak GH responses to both insulin hypoglycemia and an arginine test were significantly lowered in Groups I and II when compared to a control group of children. We conclude that only a minority of children, who previously received cranial irradiation for ALL were clinically GH deficient and, therefore, likely to benefit from GH therapy despite the finding that the majority of these children had reduced GH responses to pharmacologic stimuli.


Rickets with alopecia: an inborn error of vitamin D metabolism. Rickets with alopecia, an inborn error of vitamin D metabolism, is described in two sisters. The rachitic disorder began during the first year of life and was refractory to 50,000 IU of vitamin D2/day. Surprisingly, both children had marked elevations in serum concentrations of 1,25-(OH)2D. Although the molecular basis for this disorder is not evident to date, intestinal end-organ unresponsiveness to exceedingly high levels of 1,25-(OH)2D was present, in addition to hyporesponsiveness of bone to these high levels of the hormone, since normocalcemia was maintained despite elevated serum levels of PTH. Therapy with oral 1,25-(OH)2D3 failed to reverse the disorder, but oral phosphorus supplements resulted in significant radiographic and clinical improvement.