Regulation of lipolysis by human adipose tissue in hyperthyroidism. The effects of noradrenaline (NA) and isopropyl-noradrenaline (ISNA) on glycerol release and cAMP levels in sc adipose tissue were studied in vitro in 27 patients with hyperthyroidism. In 11 patients, the studies were repeated after 6--12 months of treatment for hyperthyroidism. A third group comprised 21 euthyroid patients otherwise healthy except for morbid obesity. The lipolytic response to ISNA, observed in untreated thyrotoxic patients, was found to be reduced by 30% when the patients were reexamined after treatment for thyrotoxicosis. This reduction was attributable to a decrease in the cAMP level. This was observed whether adipose tissue was incubated in the presence or absence of a phosphodiesterase inhibitor, theophylline. Both NA and ISNA induced 50% more rapid glycerol release and 4 times higher cAMP levels in adipose tissue of the thyrotoxic subjects than in the obese euthyroid patients. A positive correlation between tissue cAMP and glycerol release, on one hand, and mean fat cell size, on the other hand, was observed in treated thyrotoxic patients and obese euthyroid patients but was not recorded in the untreated hyperthyroid patients. The basal rate of lipolysis was not altered in thyrotoxicosis. The results suggest that the enhanced lipolytic response to catecholamines in adipose tissue of hyperthyroid patients is due to increased beta-adrenergic responsiveness. In addition, a disruption in subsequent stages of the regulatory pathway at the level of protein kinase or hormone-sensitive lipase also seems possible.


Inhibition of human chorionic gonadotropin-induced progesterone synthesis by estradiol in isolated human luteal cells. The purpose of this study was to determine whether estrogens exerted a direct inhibitory effect on progesterone synthesis in isolated human luteal cells in vitro. It was found that hCG stimulated progesterone synthesis by luteal cells, obtained from corpora lutea of the menstrual cycle, whereas cells isolated from corpora lutea of pregnancy were unresponsive to exogenous hCG. Estradiol markedly inhibited (P less than 0.001) this hCG effect in luteal cells of the menstrual cycle, and this inhibition was dose dependent. Estradiol did not block the stimulation of cAMP accumulated by hCG in the luteal cells of the cycle but did inhibit the stimulatory effect of dibutyryl cAMP on progesterone synthesis. These data suggest that estrogens may directly cause functional luteolysis in the human and that its site of action may be after the accumulation of cAMP.


Adrenocorticotropin-stimulated secretion of aldosterone and cortisol, computed from plasma and red blood cell measurements. The dynamic response of the adrenal cortex to ACTH infusion is analyzed by simulating the distribution, binding, and metabolism of cortisol and aldosterone in a multicompartmental model. The model includes the effects of temperature and cortisol concentration on aldosterone binding in plasma and the distribution between plasma and red blood cells, as verified by new observations. The secretion rates of cortisol and aldosterone were computed from serial measurements of plasma concentrations of endogenous steroids and infused tracers. The model was validated by observations after iv injection of a bolus of cortisol. Nineteen normal volunteers were studied on the fourth day on a diet containing 10 meq sodium. Endogenous ACTH was suppressed by dexamethasone, and alpha-1-24ACTH was infused at two different rates in various sequences over a 4-h period. During each hour of constant ACTH infusion, plasma cortisol continued to increase, while plasma aldosterone rose quickly, reaching a plateau within 20--30 min. Cortisol secretion approached a maximim rate after 20--30 min of ACTH infusion; the continued increase of plasma cortisol resulted from the slow equilibrium with other compartments. Aldosterone secretion rose quickly to a peak and then declined to a lower level after 20 min of ACTH infusion; the lower rate of secretion was maintained for the duration of the constant infusion of ACTH, falling abruptly within a few minutes after stopping the infusion. The characteristic differences in plasma steroid responses to various sequences of ACTH infusions can be explained by the more rapid changes in aldosterone secretion and the different clearance rates of cortisol and aldosterone, which vary with plasma cortisol concentration. The temperature at which blood is separated significantly affects plasma aldosterone measurements.


Increased somatomedin receptor sites in newborn circulating mononuclear cells. To evaluate the potential role of somatomedin as a growth factor in fetal development, we studied somatomedin receptors on circulating mononuclear cells from placental cord blood from 13 normal term newborns. Twelve healthy young adults served as controls. By radioreceptor assay of acid-chromatographed plasma, total somatomedin peptide content of cord blood was 0.30 +/- 0.02 U/ml (mean +/- SEM) compared to 1.12 +/- 0.08 U/ml for adults. Specific binding of [125I]somatomedin C to 50 X 10(6) mononuclear cells was 11.38 +/- 0.74% for newborns and 6.67 +/- 0.51% for adults. This increase in specific binding appears to be due to an increased number of receptor sites per cell. These data support a hypothesis of increased fetal sensitivity to somatomedin.


6 beta-Hydroxycortisol excretion in hypercortisolemic states. Urinary 6beta-hydroxycortisol (6betaOHF) excretion was measured and compared with free cortisol and 17-hydroxycorticosteroid (17OH) excretion in normal children, patients with Cushing's syndrome or disease (CSD), and patients during cortisol therapy. Normal 6betaOHF excretion in children was 0.23 +/- 0.03 mg/m2/24 h (mean +/- SE). No sex difference was found. ACTH infusion (40 U/day for 5 days) and high dose cortisol altered the 6 betaOHF:17OH ratio so that it was indistinguishable from the ratio seen in CSD. The fact that both Cushing's disease and high dose cortisol therapy caused the same change in the 6 betaOHF:17OH ratio suggests that cortisol and not ACTH induced 6beta-hydroxylase in hypercortisolemic subjects. Since the 6betaOHF:17OH ratio in CSD patients was always well above the normal range, measurement of 6betaOHF excretion was a better and more rapid test for chronic hypercortisolemia than urinary 17OH or free cortisol. Thus, measurement of urinary 6betaOHF is suggested as a good diagnostic test for hypercortisolemic states.


Predicting the response of growth hormone-deficient children to long term treatment with human growth hormone. A previous study showed that when GH-deficient children below the third percentile in height are treated with 0.168 U human GH (hGH)/kg BW3/4 for 10 days, their height increases by 0.3--1.9 cm during the next 8 weeks. The present study determined whether this acute response would predict the child's long term response to 1 yr of treatment with the same dose of hGH given three times a week. Eighteen GH-deficient children and adolescents, aged 8--16 yr, were measured every 2 weeks over 108 weeks. After a control period of 12 weeks (period 1), the patient received hGH for 10 days. During the remainder of the 12 weeks of period 2 and during the next 12 weeks (period 3), hGH was not given. Patients recieved hGH three times a week during periods 4 and 5 (24 weeks each). Periods 6 and 7 (12 weeks each) were posttreatment control periods. During periods 1, 3, 6, and 7, rate of growth was less than 0.2 cm/month. During period 2, the rate ranged between 0.1--0.8 cm/month. During periods 4 and 5, the growth rate ranged from 0.2--1.0 cm/month. Rate of growth during periods 4 and 5 (y) was related to rate during period 2 (x) by the equation y = 0.027 + 1.17 x. The correlation coefficient between y and x was 0.91 (P less than 0.001). The increment in height which will occur during 48 weeks of treatment can be predicted from the response to 10 days of treatment by this equation. The SE of the prediction averages +/- 1.2 cm/yr.


Beta-Adrenergic receptors of human lymphocytes are unaltered by hyperthyroidism. Lymphocytes from 12 patients with untreated hyperthyroidism were compared to lymphocytes from age- and sex-matched euthyroid control subjects to test the hypothesis that alterations in beta-adrenergic response mechanisms occur in human hyperthyroidism. The binding of (-)[3H]dihydroalprenolol, a compound previously shown in these cells to label binding sites having the characteristics of beta-adrenergic receptors, was assayed and no significant difference was found between the two groups. In addition, the accumulation of cAMP in response to isoproterenol was determined by RIA and, again, no difference was found.


Virilizing adrenal adenoma stimulated by dexamethasone in a middle-aged woman. In a middle-aged woman with virilizing adenoma, 2 mg dexamethasone increased urinary excretion of 17-ketosteroids (17-KS) and 17-hydroxycorticosteroids, whereas 8 mg dexamethasone increased urinary excretion only of 17-KS. With discontinuation of dexamethasone, 17-KS excretion returned to the predexamethasone level. Dexamethasone depressed the basal level of cAMP synthesis and basal testosterone production by the normal adrenal tissue in vitro. Dexamethasone also depressed the increase of cAMP produced by ACTH in the normal tissue. In contrast, dexamethasone increased basal cAMP synthesis and stimulated testosterone secretion in the tumor tissue. ACTH and dexamethasone were additive in their effects on cAMP and testosterone in the tumor tissue. It is suggested that dexamethasone acted directly on the adrenal tumor to stimulate steroid secretion in this patients.


Epidermal growth factor (urogastrone) in human tissues. Human epidermal growth factor (hEGF), which stimulates the growth of a variety of tissues, was first isolated from mouse submandibular glands, but is also excreted in large amounts (about 50 micrograms/day) in human urine and is probably identical to human beta-urogastrone (hUG), a potent inhibitor of stimulated gastric acid secretion. However, the primary tissue source of hEGF/hUG is as yet unknown. The hEGF/hUG in homogenates of human salivary glands and a wide variety of other endocrine and nonendocrine tissues was extracted by Amberlite CG-50 cation exchange chromatography and immune affinity chromatography using the immunoglobulin fraction of rabbit anti-hEGF serum covalently bound to agarose. The extracts were subjected to homologous hEGF RIA. Immunoreactive hEGF was found in extracts of adult submandibular gland, thyroid gland, duodenum, jejunum, and kidney, but not in several fetal tissues. The tissue immunoreactive hEGF was similar to standard hEGF in terms of immunoreactivity and elution from Sephadex G-50 Fine resin, but its concentrations were very low (1.3-5.5 ng/g wet tissue). Thus, it is not certain that these tissues represent the only source of the large amounts of hEGF/hUG that appear to be filtered by the kidneys each day.


Circadian cortisol secretory rhythms in Cushing's disease. Plasma cortisol was measured at 20-min intervals for 24 h in eight patients with Cushing's disease and ACTH-secreting pituitary tumors. The 24-h mean (+/- SD) cortisol level was 25.6 +/- 11.3 microgram/dl (range, 15.5--40.6), which was significantly higher than the level of normal control subjects (P less than 0.01). The 24-h mean ACTH level varied between 22--107 pg/ml, with a mean +/- SD of 63.4 +/- 27.2. The mean ACTH level was higher than that of the control subjects but the difference was not statistically significant. The 24-h cortisol secretory pattern was characterized by an absence of the normal circadian variation and a failure of the plasma cortisol level to fall to less than 2 microgram/dl between 2300--0300 h. The coefficient of variation, an expression of the amplitude of cortisol secretory episodes, was significantly decreased in patients with Cushing's disease compared to normal control subjects; there was no significant difference in the number of cortisol secretory episodes in the patients vs. control subjects. Three of the patients were restudied after successful resection of their ACTH-secreting pituitary tumors. Two showed normalization of their 24-h circadian cortisol patterns and normal metyrapone responses. In the third, the 24-h mean cortisol level was normal, but the circadian cortisol rhythm remained abnormal. This patient had diminished ACTH reserve, demonstrated by a subnormal response to metyrapone. Additional studies will be required to determine if normalization of the circadian cortisol rhythm occurs in all patients with Cushing's disease who are cured after transsphenoidal microsurgery and who also show normal ACTH reserve.


Direct inhibitory effect of dexamethasone on steroidogenesis of human adrenal in vivo. A rapid ACTH test was used to investigate the direct effect of glucocorticoid on the steroidogenesis by the adrenal cortex in man. The plasma cortisol response to 250 microgram iv synthetic alpha-ACTH-(1--24) (Cortrosyn) was determined in normal subjects pretreated with 2 and 12 mg dexamethasone. The 15- and 30-min increments in plasma cortisol levels in response to ACTH injection after pretreatment with 12 mg dexamethasone were significantly suppressed compared to values obtained after pretreatment with 2 mg dexamethasone. The present study suggests that there is a direct inhibitory effect of glucocorticoid on adrenocortical steroidogenesis in man.


Hemodialysis-resistant hypertension: control with an orally active inhibitor of angiotensin-converting enzyme. In two patients with end stage renal disease and dialysis-resistant hypertension, the orally active inhibitor of angiotensin-converting enzyme, captopril (SQ14,225; 2-D-methyl-3-mercaptopropranoyl-L-proline, dramatically lowered blood pressure both before and during dialysis. This agent holds promise as an alternate to bilateral nephrectomy in such patients.


Lack of a diurnal plasma adenosine 3',5'-monophosphate rhythm. Plasma cAMP levels were measured using a protein-binding radioassay in nine healthy volunteer subjects over a period of 24 h under standardized conditions and also after an hour of orthostatism. No circadian variations of cAMP in plasma were observed. After orthostatism, however, there was a significant rise in cAMP concentrations.


Phentolamine and the action of somatostatin in man. The inhibition by somatostatin (SRIF) of basal and arginine-stimulated glucagon, insulin, and glucose levels was compared with that obtained when SRIF was preceded by alpha-adrenergic blockade with phentolamine. No noteworthy differences were observed, except that the characteristic rebound of insulin upon discontinuation of SRIF was significantly lower with phentolamine (P less than 0.01). These results indicate that inhibition of basal and arginine-stimulated glucagon and insulin secretion by SRIF in man is not mediated through activation by alpha-adrenergic receptors.


Regulatory role of triiodothyronine in the degradation of low density lipoprotein by cultured human skin fibroblasts. Degradation of [125I]-labeled low density lipoprotein (LDL) by cultured normal human skin fibroblasts was enhanced by exposure to concentrations of L-triiodothyronine (T3) spanning the physiological range. Analysis of LDL concentration curves suggests that T3 may increase LDL receptor number rather than alter the affinity of LDL for its receptor. No effect of T3 on LDL degradation was observed in receptor negative cells. Thus thyroid hormone appears to regulate receptor-mediated LDL degradation by increasing the binding of the lipoprotein to its cell-surface receptor.


Sensitive solid-phase radioimmunoassay for detection of human immunoglobulin G antibodies to varicella-zoster virus. A sensitive solid-phase radioimmunoassay for detection of antibodies to varicella-zoster virus (VZV) is described. The antigen consisted of a sonically disrupted extract of VZV-infected human embryo cells. 125I-labeled rabbit anti-human immunoglobulin G (IgG) specific for the Fc portion of human IgG was used to detect human IgG bound to viral antigen. With this technique, 193 human sera were evaluated for their IgG antibody titer against ZVZ. Subjects included 62 healthy adults, 33 young children (12 healthy), and 49 patients. Titers obtained by the radioimmunoassay were compared with those obtained by indirect fluoresence antibody staining of membrane antigen. The radioimmunoassay technique described gave titers approximately 5 X 10(4) times higher than those shown by indirect fluorescence. It can be used for routine diagnosis, but is especially suited to determining immune status to VZV, as defined by presence or absence of antibodies to the virus; for epidemiological studies; or for determining patients at risk who are exposed to the virus. No heterotypic titer rises to VZV were observed in sera with fourfold or greater rises to Epstein-Barr virus or cytomegalovirus. Sera of eight subjects with fourfold or greater titer rises to herpes simplex virus reacted in various ways: in six cases no significant change occurred in titer to VZV; one had a significant decrease in titer by the radioimmunoassay; and one had a significant increase. Possible reasons for these titer changes are discussed.


Serodiagnosis of viral hepatitis A by a modified competitive binding radioimmunoassay for immunoglobulin M anti-hepatitis A virus. A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluated and compared with a standard solid-phase radioimmunoassay for anti-HAV, CBA was found to be sensitive and specific for the detection of anti-HAV, as demonstrated by the 98% concordance of CBA and solid-phase radioimmunoassay test results. The standard CBA test was modified for the differential detection of acute (immunoglobulin M) and convalescent (immunoglobulin G) anti-HAV by incorporation of a step in which immunoglobulin G anti-HAV was preferentially absorbed with S. aureus cells (protein A). The modified CBA test was shown to be capable of differentiating between acute- and convalescent-phase sera. The modified CBAM test was able to detect immunoglobulin M anti-HAV up to approximately 4 weeks after the onset of illness.


Antibody reactivity to human and vervet cytomegalovirus early antigen(s) in sera from patients with active cytomegalovirus infections and from asymptomatic donors. Human fibroblasts were infected with vervet cytomegalovirus (VCMV) and cultured in medium containing 50 micrograms of cytosine arabinoside per ml for 72 h. Early antigens (EAg) were detected in the nuclei of infected cells by an indirect fluorescent antibody test with human sera having antibody to EAg of human cytomegalovirus (HCMV). Sera from patients with serological and/or virological evidence of active HCMV infection and from asymptomatic blood donors were examined for antibody activity with the HCMV and VCMV EAg's. The HCMV and VCMV EAg's were comparable in detecting levels of antibody activity and fluctuations in antibody titer of paired sera from patients. A total of 81% of patient sera reactive with HCMV EAg were also reactive with VCMV EAg. In contrast, only 14% of the asymptomatic donor sera reactive with HCMV EAg were also reactive with VCMV EAg. The VCMV EAg, therefore, appeared to differentiate latent from active infections in humans more effectively than did HCMV EAg.


Comparison of occurrence of antibodies to human cytomegalovirus as demonstrated by immunofluorescence and indirect hemagglutination techniques. Indirect immunofluorescence and hemagglutination study of reactivity to human cytomegalovirus immediate early, early, and late antigens revealed that antibody responses to these antigens varies greatly and that hemagglutination is positive when sera contain only antibody to one or both early antigens.


Feline leukemia virus: survival under home and laboratory conditions. Feline leukemia virus maintained its infectiousness in cell culture medium at 37 degrees C or lower for at least 48 h. However, virus in saliva or medium was inactivated within a few hours if allowed to dry.


Persistence of antibodies to rotavirus in human milk. Human milk obtained from 21 American nursing mothers was studied for the presence of secretory immunoglobulin A antibody to rotavirus, the most common etiological agent of infantile gastroenteritis. Antibody was quantitated by adaptation of a recently described solid-phase radioimmunoassay technique that employs simian rotavirus as a convenient substitute antigen for human rotavirus. Of the mothers tested, 80% (12 of 15) possessed milk antibody within a week of parturition, whereas 56% of those tested (5 of 9) secreted milk antibody as late as 6 or 9 months postpartum. Specificity of the radioimmunoassay was demonstrated by absorption of antibody with purified rotavirus. Our detection by radioimmunoassay of antibody to rotavirus in milk samples collected past the colostrum stage is in contrast to other studies that have failed to detect antibody in human milk by immunofluorescence or neutralization tests. The present study also suggested that the appearance of secretory immunoglobulin A antibody in the milk of mothers previously lacking milk antibody may be correlated with subclinical infection of the mother with rotavirus.


Canine kidney cell line for isolation of respiratory viruses. By means of a continuous canine kidney cell line (MDCK), influenza viruses were rapidly isolated from specimens collected from patients with respiratory disease. The cell line proved more sensitive than either eggs or rhesus monkey cells for currently circulating influenza A and B strains. Influenza viruses caused a distinct cytopathology within 5 days of inoculation if trypsin-ethylenediamine-tetraacetic acid was incorporated into the medium. Sufficient hemagglutinin was produced on the initial tissue culture passage to allow direct identification of isolates by hemagglutinin inhibition tests. A variety of other respiratory viruses replicated in MDCK, and over a 10-month period 211 of 600 specimens (35%) yielded viruses.


Effect of blood dilution on recovery of organisms from clinical blood cultures in medium containing sodium polyanethol sulfonate. This clinical study was designed to evaluate the standard laboratory protocol that requires blood specimens be diluted with greater than or equal to 10 volumes of media. Blood was collected from hospitalized patients, and 1 ml was inoculated into each of three vials containing 2.3, 7.3, and 24 ml of BACTEC 6B aerobic medium resulting in dilutions of 1:4, 1:10, and 1:30, respectively. The three test vials were treated identically, and the study was carried out at four hospitals. Of the 2,550 sets of vials inoculated, 174 were positive with clinically significant isolates from 105 patients. There was no difference in the number of positive cultures recovered by 24 h (67%) or 48 h (90%) from any dilution. These percentages agreed with other reports from BACTEC users. The number of positive vials (139, 144, 147, respectively) at each dilution was not significantly different, indicating that all three dilutions showed equal recovery of pathogenic microorganisms. Despite this overall equality, two patients, one on antibiotic therapy, were found to have correlated cultures which failed to grow at the 1:4 dilution. This finding implies that a 1:4 dilution of blood cannot be recommended unequivocably despite the higher overall recovery rate of positive cultures.


Complement-fixing immunoglobulin M antibody response in patients with infantile gastroenteritis. Complement-fixing immunoglobulin M antibody to infantile gastroenteritis virus (a rotavirus) was detected with highest sensitivity when the antibody-antigen-complement mixture was incubated at 37 degrees C for 1 h prior to the addition of sensitized sheep erythrocytes. Sucrose gradient centrifugation of sera collected sequentially from four patients after infection detected 19S complement-fixing antibody up to 5 weeks, with highest titers at 1 week, after the onset of illness. Treatment of the whole sera with 2-mercaptoethanol decreased complement-fixing titers only up to 2 weeks after onset of illness.


Antiherpesvirus activity in human sera and urines after administration of adenine arabinoside: in vitro and in vivo synergy of adenine arabinoside and arabinosylhypoxanthine in combination. The minimum inhibitory concentration (MIC) of adenine arabinoside (ara-A) in rabbit kidney microtiter tissue cultures (RK-13) to a prototype strain of herpes simplex virus, type 1 (E115) based upon inhibition of cytopathic effects is 1.5 mug/ml. In this system, the MIC of arabinosylhypoxanthine (ara-Hx), the major in vivo metabolic derivative of ara-A, is 75 mug/ml. Inhibition of cytopathic effects of herpes simplex virus, type 1 (HSV-1) in microtiter wells of RK-13 cells varies directly with the concentrations of ara-A or ara-Hx, and inversely with residual HSV-1. The MIC of ara-A for HSV-1 in RK-13 cells is 5-20 times lower than similar measures with vero renal, mouse embryo, or human foreskin cultures. With RK-13 tissue cultures in microtiter plates, an assay for "ara-A equivalents" in human body fluids was developed which compares in sensitivity with high pressure liquid chromatography and has the advantage of simultaneously measuring combined antiherpesvirus effects of ara-A and its major metabolic derivative, ara-Hx. In vitro checkerboard studies in RK-13 cells confirmed that ara-A and ara-Hx in combination had antiviral effects which are synergistic. The total of the fractional MIC of ara-A plus ara-Hx in combination also varies inversely with residual HSV-1 in microtiter wells. Because virus adsorption is complete at 2 h before specimens to be tested are added in this assay, and because human interferon is not measured in rabbit cells, the antiviral assay is not affected by the presence of type-specific antiherpesvirus antibody or human interferon.Antiviral activity (AVA) was assayed as ara-A equivalents in sera and urines from 10 patients with serious herpesvirus infections who received 2.5-20 mg/kg daily of ara-A by intramuscular or intravenous routes. When a dosage schedule of 10 mg/kg per day or more was used, sustained concentrations of AVA that ranged from 0.8 to 14.4 mug/ml were found. When an inhibitor of adenosine deaminase (covidarabine) was not added to the specimens, mean serum concentrations were congruent with3.0 mug/ml (10 mg/kg per day, i.v.), and 4.1 mug/ml (20 mg/kg per day). However, in a single patient given 20 mg/kg of ara-A daily with covidarabine immediately added to the sera, the mean concentration of AVA was 12.9 mug/ml. Urines contained even higher AVA. Assays of 19 sera were performed both by microbiologic assay for AVA and by high pressure liquid chromatography for ara-A and ara-Hx. AVA was greater by microbiologic assay, and was greater than that which could be accounted for by stoichiometric chromatographic measures of ara-A and ara-Hx. These results with sera of treated patients are consistent both with the in vitro synergy of ara-A and ara-Hx found by checkerboard titrations, and with the beneficial responses to ara-A of patients with herpesvirus infections reported here and elsewhere.


Effects of osmolality and oxygen availability on soluble cyclic AMP-dependent protein kinase activity of rat renal inner medulla. The renal inner medulla is ordinarily exposed to osmolalities that are much higher and to O2 tensions that are lower than those in other tissues. The effects of media osmolality and O2 availability on basal and arginine vasopressin(AVP)-responsive soluble cyclic (c)AMP-dependent protein kinase activity were examined in slices of rat inner medulla. Increasing total media osmolality from 305 to 750 or 1,650 mosM by addition of urea plas NaCl to standard Krebs-Ringer bicarbonate buffer significantly reduced basal cAMP content and protein kinase activity ratios. This occurred in the presence or absence of O2. Incubation of slices in high osmolality buffer also blunted increases in inner medullary slice cAMP and protein kinase activity ratios induced by O2. These changes reflected predominantly an action of the urea rather than the NaCl content of high osmolality buffers. In contrast to effects on basal activity, high media osmolality significantly enhanced activation of inner medullary protein kinase by AVP. Conversely, increases in media O2 content suppressed AVP stimulation of enzyme activity. This inhibitory effect of O2 was best expressed at low osmolality. Naproxen and ibuprofen, inhibitors of prostaglandin biosynthesis, reduced basal kinase activity ratios and increased AVP responsiveness in the presence, but not in the absence, of O2. Exogenous prostaglandins (PG) modestly increased (PGE2 and PGE1) or did not change (PGF2alpha) cAMP and protein kinase activity ratios in O2-deprived inner medullary slices. Protein kinase activation by PGE2 was not observed in oxygenated inner medulla with high basal activity ratios. The stimulatory effects of PGE2 and PGE1 on protein kinase activity observed in O2-deprived slices were additive with those of submaximal or maximal AVP. PGE2, PGE1, and PGF2alpha all failed to suppress AVP activation of protein kinase. Thus, enhanced endogenous PGE production may contribute to the higher basal protein kinase activity ratios induced by O2. However, the results do not support a role for PGE2, PGE1, or PGF2alpha in O2-mediated inhibition of AVP responsiveness. The present data indicate that both solute content and O2 availability can alter the expression of AVP action on cAMP-dependent protein kinase activity in inner medulla. AVP activation of protein kinase is best expressed when osmolality is high and O2 availability is low, conditions that pertain in inner medulla during hydropenia.


Altered activity of the nucleotide regulatory site in the parathyroid hormone-sensitive adenylate cyclase from the renal cortex of a patient with pseudohypoparathyroidism. A series of clinical studies suggest that the primary defect underlying pseudohypoparathyroidism is an abnormality of the parathyroid hormone-receptor-adenylate cyclase complex of the renal cortical cell plasma membrane. In the present study we compared parathyroid hormone-stimulated adenylate cyclase activity in membrane preparations from the renal cortex of three controls and a patient with pseudohypoparathyroidism. In the pseudohypoparathyroid preparation the Km for ATP was significantly greater and parathyroid hormone elicited markedly diminished adenylate cyclase activity at a subsaturating concentration of ATP. In contrast, the dose-response effect of enzyme activity to parathyroid hormone was the same in the control preparations, and that of the pseudohypoparathyroidism kidney, at a saturating concentration of ATP. The apparent alteration in enzyme kinetics, however, was normalized upon addition of guanosine 5'-triphosphate to the reaction mixtures. These results indicate that the defect in the parathyroid hormone-receptor-adenylate cyclase complex of the renal cell membranes, in our patient with pseudohypoparathyroidism, is an abnormal nucleotide receptor site of decreased activity. Such a defect may result in partial uncoupling of the parathyroid hormone receptor and adenylate cyclase, rendering the organ refractory to hormonal stimulation.


Viral infection-homograft interactions in a murine model. The effects on some host defenses of murine cytomegalovirus (MCMV) and(or) EL(4), a mouse ascites homograft, were studied in mice. Assays of cellular and humoral immunity in response to either or both of these perturbations were carried out by quantitation of various immune activities.Limited studies demonstrated no effect of EL(4) inoculation on the host response to MCMV by organ viral titer, duration of viral persistence, or anti MCMV complement-fixing antibody titer. Prior infection with MCMV, however, resulted in greatly reduced numbers of splenocytes, the source in this study of immune effector cells. Residual splenocytes demonstrated less response to both phyto-hemagglutinin and lipopolysaccharide, particularly in the 2-3-wk interval after infection. Similarly, responder cells in mixed lymphocyte cultures were less reactive when derived from infected animals. Lymphocyte-mediated cytolysis of EL(4) was significantly less in mice infected on the day of and 7, 14, and 21 days before the tumor homograft with a return to control levels by 28 days. 90% of the cell-mediated cytolysis could be eliminated by treatment with anti-theta serum. Serum-mediated cytolysis of EL(4) was also reduced in infected animals. No suppressor cells or serum inhibitory factors could be identified in infected animals. Although alternative explanations exist, this study suggests that in infected animals there is a significant reduction in both the number and function of bone marrow-derived and thymus-derived cells directed against the alloantigens in EL(4).


Modulation of cyclic nucleotides in islated rat glomeruli: role of histamine, carbamylcholine, parathyroid hormone, and angiotensin-II. Because glomerular functions are modulated by numerous humoral agents, probably acting through cyclic nucleotides, the effects of some polypeptide hormones and biogenic amines on cyclic AMP (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) were studied in glomeruli isolated from rat renal cortex. Glomeruli and cortical tubules were prepared by a combination of sieving and density-gradient centrifugation. Under basal conditions, the contents of cAMP and cGMP in glomeruli were significantly higher than in tubules and unfractionated renal cortical tissue.Histamine caused a striking increase in cAMP in glomeruli (+Delta% 675+/-87) and, to a lesser degree, increased cAMP in tubules (+Delta% 103+/-25) or in tissue slices. This stimulation was dose-dependent in the range of 1 muM-1 mM histamine. Metiamide (an H(2)-antagonist), but not pyrilamine (an H(1)-antagonist) blocked the effect of histamine on cAMP, which indicates that histamine causes its effect via interaction with H(2) receptors. Histamine caused less extensive increases in cGMP in both glomeruli and tubules. Carbamylcholine caused a marked increase in cGMP in glomeruli (+Delta 295+/-7) and a much lower increase in tubules (+Delta% 70+/-20); these effects were blocked by atropine. Parathyroid hormone (1 mug/ml) increased cAMP and, to a much lesser degree, also cGMP in glomeruli. In tubules, parathyroid hormone caused much more extensive increases in cAMP than in glomeruli; no changes, or rather a small decline in cGMP, was observed. Angiotensin-II (2 muM) markedly lowered cAMP in glomeruli (-Delta% -45+/-8) and in tubules (-Delta% 33+/-7) but had no effect on cGMP. Bradykinin (20 muM) did not consistently influence either cAMP or cGMP in glomeruli or tubules. Present results demonstrate that cAMP and cGMP metabolism in glomeruli are controlled independently by humoral agents known to alter glomerular functions in vivo. Our findings are consistent with the view that histamine and cholinergic agents generated and (or) released locally in glomeruli or in their vicinity may play important roles as mediators of immunopathological injury of glomeruli, and that these effects are mediated by cAMP and (or) cGMP through interaction with H(2) receptors and muscarinic receptors. Likewise, our results suggest that the effects of angiotensin-II and parathyroid hormone on glomerular dynamics may be mediated by cyclic nucleotides.Thus, we surmise that extrarenal as well as intrarenal humoral agents may play an important role in the pathology and physiology of glomeruli through mediation of either cAMP, cGMP, or both.


Studies on the structure of low density lipoproteins isolated from Macaca fascicularis fed an atherogenic diet. Cynomolgus monkeys, Macaca fascicularis, fed cholesterol-containing saturated-fat diets develop increased levels of high molecular weight plasma low density lipoproteins (LDL), associated with accelerated atherosclerosis. To study the composition and structure of these abnormal particles, LDL from monkeys, fed atherogenic and control diets, were characterized chemically and examined by differential scanning calorimetry and low-angle X-ray scattering. LDL from animals on the experimental diet showed an increase in molecular weight (4.0 to 7.0 x 10(6), experimental diet compared with 3.0 to 3.7 x 10(6), control diet) associated with a large increase in cholesterol ester content and concomitant smaller increases in protein, phospholipid, and free cholesterol. There was a strong positive correlation between molecular weight and the number of saturated and monounsaturated cholesterol esters in the particle. In contrast, particle content of polyunsaturated cholesterol esters remained constant despite large changes in total particle cholesterol esters.When examined by calorimetry and X-ray scattering, LDL from monkeys on both diets diplayed a reversible transition of cholesterol esters from an ordered smeticlike (layered) structure to a more disordered state. For all animals on the experimental diet, the peak temperature of the cholesterol-ester transition (42-48 degrees C) was above body temperature (39 degrees C), but below body temperature on the control diet (34-38.5 degrees C). In the experimental group, the transition temperature was correlated with the LDL molecular weight. However, after thermal disruption of LDL, liquid-crystalline transitions of LDL cholesterol esters were observed in the same temperature range as in the intact lipoprotein, which shows that changes in particle size had little effect on the cholesterol-ester transition temperature. Rather, the transition temperature was determined by the degree of saturation of the LDL cholesterol ester fatty acids and the LDL cholesterol ester: triglyceride ratio, both of which correlated with increased LDL molecular weight.The existence of smectic-like cholesterol ester in LDL at body temperature was clearly a discriminating feature between monkeys on control and experimental diets. Diet-induced changes in the lipid composition of precursor lipoproteins of LDL appeared to lead to the existence of smectic-like cholesterol ester in LDL above body temperature. The altered composition and structure of the core lipids of high molecular weight LDL probably account, in part, for the previously documented correlation between increased LDL molecular weight and atherosclerosis in this species.


Presence of immunoreactive beta-endorphin in normal human plasma: a concomitant release of beta-endorphin with adrenocorticotropin after metyrapone administration. To elucidate whether or not beta-endorphin exists in plasma of normal subjects, plasma extracts obtained before and after metyrapone administration were subjected to gel exclusion chromatography, and fractions obtained were assayed by a sensitive radioimmunoassay for beta-endorphin. The basal plasma level of beta-endorphin was 5.8 +/- 1.1 pg/ml (mean +/- SE, n = 5), which rose significantly to the level of 48.9 +/- 3.8 pg/ml after a single oral dose (30 mg/kg of body wt) of metyrapone administration (P less than 0.001). Plasma ACTH levels also increased from the mean basal level of 73 +/- 4 pg/ml to 269 +/- 41 pg/ml after metyrapone administration. These results indicate that beta-endorphin, distinct from beta-lipotropin, exists in normal human plasma and that it is released from the pituitary concomitantly with ACTH.


Myeloperoxidase-mediated platelet release reaction. The ability of the neutrophil myeloperoxidase-hydrogen peroxide-halide system to induce the release of human platelet constituents was examined. Both lytic and nonlytic effects on platelets were assessed by comparison of the simultaneously measured release of a dense-granule marker, [(3)H]serotonin, and a cytoplasmic marker, [(14)C]adenine. Incubation of platelets with H(2)O(2) alone (20 muM H(2)O(2) for 10 min) resulted in a small, although significant, release of both serotonin and adenine, suggesting some platelet lysis. Substantial release of these markers was observed only with increased H(2)O(2) concentrations (>0.1 mM) or prolonged incubation (1-2 h). Serotonin release by H(2)O(2) was markedly enhanced by the addition of myeloperoxidase and a halide. Under these conditions, there was a predominance of release of serotonin (50%) vs. adenine (13%), suggesting, in part, a nonlytic mechanism. Serotonin release by the complete peroxidase system was rapid, reaching maximal levels in 2-5 min, and was active at H(2)O(2) concentrations as low as 10 muM. It was blocked by agents which inhibit peroxidase (azide, cyanide), degrade H(2)O(2) (catalase), chelate Mg(2+) (EDTA, but not EGTA), or inhibit platelet metabolic activity (dinitrophenol, deoxyglucose).These results suggest that the myeloperoxidase system initiates the release of platelet constituents primarily by a nonlytic process analogous to the platelet release reaction. Because components of the peroxidase system (myeloperoxidase, H(2)O(2)) are secreted by activated neutrophils, the reactions described here may have implications for neutrophilplatelet interaction in sites of thrombus formation.


The influence of hyperthyroidism and hypothyroidism on the beta-adrenergic responsiveness of the turkey erythrocyte. The mechanisms responsible for altered adrenergic tone in hyperthyroidism and hypothyroidism are not fully understood. To investigate these mechanisms, the beta-adrenergic receptor-cyclic AMP complex of the turkey erythrocyte was studied among groups of normal, hyperthyroid, and hypothyroid turkeys. In erythrocytes obtained from hypothyroid turkeys, there were fewer beta-adrenergic receptors than in normal cells as determined by the specific binding of [(125)I]iodohydroxybenzylpindolol, as well as associated decreases both in catecholamine-responsive adenylate cyclase activity and in cellular cyclic AMP content. In contrast, erythrocytes obtained from hyperthyroid turkeys contained the same number of beta-receptors and had the same catecholamine-responsive adenylate cyclase activity as cells from normal birds. Other characteristics of the beta-receptors in cells from hyperthyroid birds were indistinguishable from those present in normal erythrocytes. However, within the range of circulating catecholamine concentrations, 5-50 nM, the erythrocytes of the hyperthyroid turkeys generated substantially more cyclic AMP after exposure to isoproterenol than did normal cells. These results suggest that thyroid hormone affects beta-receptor-cyclic AMP interrelationships in the turkey erythrocyte by two distinct mechanisms: (a) In hypothyroidism, both beta-receptors and catecholamine-dependent cyclic AMP formation are coordinately decreased; (b) in hyperthyroidism, beta-receptors are unchanged but there is an amplification of the hormonal signal so that occupation of a given number of receptors at physiological concentrations of catecholamines leads to increased levels of cyclic AMP.


Differential sensitivity of lymphocyte subpopulations to suppression by low density lipoprotein inhibitor, an immunoregulatory human serum low density lipoprotein. Reports by a number of investigators have described the thymus-derived (T)-cell dependence of immunoglobulin synthesis by pokeweed mitogen (PWM) stimulated human peripheral blood bone marrow-derived (B) cells. Because of the cooperative nature of this in vitro system, it was chosen for examination of the differential effects of low density lipoprotein inhibitor (LDL-In) on B- and T-cell functions. Supernates from 7-d cultures that contained either peripheral blood mononuclear cells (PBM) or combinations of isolated lymphocyte populations were assayed for immunoglobulin (Ig)G by competitive inhibition radio-immunoassay. LDL-In suppression of whole PBM IgG synthesis occurred at 5-20 mug protein/ml and was independent of PWM concentration. Maximal suppression required preincubation of cells with LDL-In before stimulation. Suppression was also observed when B cells alone were exposed for 24 h to LDL-In before PWM stimulation; these suppressed B cells were not rescued by normal T cells. Exposure of T cells alone to low doses of LDL-In for 24 h augmented, but high doses suppressed, IgG synthesis, suggesting a differential effect on T-helper vs T-suppressor cell populations. Independent LDL-In exposure of T-helper or T-suppressor cell enriched populations, separated by rosetting with IgG- or IgM-coated ox erythrocytes, identified the T-suppressor cell populations as the most sensitive of the lymphocyte populations tested. The sensitivities of lymphocyte subpopulations to LDL-In, relative to PBM, were 2.8, 1.2, and 0.3 for the T-suppressor cells, B cells and T-helper cells, respectively. Thus, both B and T lymphocytes are sensitive to and can be regulated by LDL-In. In addition, the biologic activity observed when unseparated PBM are exposed to LDL-In appears to represent a composite of the sensitivity of each of the lymphocyte subpopulations.


Differential and integral corticosteroid feedback effects on ACTH secretion in hypoadrenocorticism. Recent work suggests the existence of a dual corticosteroid feedback mechanism of stress-induced ACTH secretion in the rat. This possibility led us to study the kinetics of suppression of ACTH levels by corticosteroid administration in patients with nonstress ACTH hypersecretion secondary to hypoadrenocorticism. Cortisol was administered according to different protocols, which were chosen to provide extreme variations of the input signal. By this means, two phases of suppression of ACTH levels could be differentiated. A first decrease occurred without latency whenever, and as long as, plasma cortisol levels were rising. There was a linear regression between the logarithm of the increments in cortisol concentrations and the decrease in ACTH levels per minute (r = 0.951) (differential or rate-sensitive feedback mechanism). Neither the absolute doses of cortisol, nor plasma cortisol concentrations were closely correlated with the degree of suppression of ACTH by this rapid mechanism. A second decrease in ACTH levels began congruent with30 min after corticosteroid administration. In this case there was a significant linear regression between the degree of inhibition of ACTH levels and the cortisol doses (r = 0.997) (integral or dose-sensitive feedback mechanism). The dose-sensitive feedback effects of dexamethasone were less than might have been predicted from its relative anti-inflammatory potency. No rate-sensitive effects were seen with dexamethasone doses of 1.0 or 1.25 mg.


Interactions between vitamin D deficiency and phosphorus depletion in the rat. To evaluate the role of vitamin D in the physiologic response to phosphorus depletion (P depleton) and the response to vitamin D administration in P depletion, we studied vitamin D-deficient (-D) rats, fed either a normal or low phosphorus diet and then injected intraperitoneally on alternate days with replacement vitamin D(3), 1.25 mug qod (D(3)); 1.25-dihydroxy-vitamin D(3)[1,25(OH)(2)D(3)] in physiologic, 54 ng qod (LD), and pharmacologic doses, 400 ng qod (HD); or vehicle alone (-D). The following results were obtained: (a) With P depletion, urinary excretion of inorganic phosphorus (Pi) fell to almost undetectable levels in -D rats, and two physiologic features of P depletion a calcemic effect and hypercalciuria, ensued. (b) With administration of vitamin D(3) or 1,25(OH)(2)D(3) in either doses to P-depleted rats, the renal retention of Pi was unaltered despite a significant elevation of serum Pi. (c) The calcemic response to P depletion was accentuated by vitamin D sterols, and the hypercalciuria of P depletion was reduced by 1,25(OH)(2)D(3), HD > LD > D(3). (d) In -D animals receiving normal Pi (+P), D(3), and 1,25(OH)(2)D(3), both LD and HD produced a significant calcemic and phosphatemic effect. (e) Urinary Pi excretion in +P animals was reduced slightly by vitamin D(3) whereas 1,25(OH)(2)D(3), both LD and HD, lowered urinary Pi markedly despite an increased serum Pi. (f) The serial values of serum Ca and Pi and urinary Ca in PD rats and the sequential values for urinary and serum Pi in +P rats indicated more rapid effects of 1,25(OH)(2)D(3), both HD and LD, compared with D(3). We conclude that: (a) The renal adaptation and physiologic response to PD does not require the presence of vitamin D. (b) 1,25(OH)(2)D(3) may directly enhance the renal tubular reabsorption of Pi even as serum Pi rises. (c) A hypocalciuric action of 1,25(OH)(2)D(3) in rats on low phosphorus diet could be direct or occur as a consequence of an increase in serum Pi produced by 1,25(OH)(2)D(3). The different sequential renal response to D(3) compared with 1,25-(OH)(2)D(3) raises the possibility that other natural forms of vitamin D(3) [i.e., 25(OH)D(3), 24,25(OH)(2)D(3), etc.] which may be present in vitamin D-fed rats but not those given only 1,25(OH)(2)D(3), could modify the actions of 1,25(OH)(2)D(3).


Regulation of cytoplasmic dihydrotestosterone binding in dog prostate by 17 beta-estradiol. 17 beta-estradiol enchances androgen-induced prostate growth in the castrate dog to a degree comparable to that seen in spontaneous prostatic hypertrophy. To investigate the mechanism of this synergism, cytosol androgen binding was measured by a density gradient technique in prostates of control and 17 beta-estradiol-treated castrate dogs. [3H]Dihydrotestosterone was bound principally to a moiety that averaged 8.6S in size. Approximately twofold enhancement of this binding by 17 beta-estradiol was demonstrable after 1 wk of treatment with 750 microgram/wk and after 3 wk with 75 microgram/wk. Under conditions in which binding in the 8S region was demonstrable with dihydrotestosterone and testosterone no binding of 3 alpha-androstanediol or progesterone was detectable. Thus, enhancement by 17 beta-estradiol of a prostate cytosol androgen-binding protein occurs under circumstances in which 17 beta-estradiol enhances androgen-mediated prostatic growth.


The adrenal receptor for angiotensin II is altered in essential hypertension. To determine the mechanism underlying altered adrenal responsiveness in patients with essential hypertension, the renin-angiotensin-aldosterone axis was assessed in normotensive and hypertensive subjects using three pharmacological probes: SQ 20881, a converting enzyme inhibitor; saralasin, a competitive angiotensin antagonist with prominent agonist properties; and angiotensin itself. All subjects were studied while supine and in balance on a 10 meq Na/100 meq K intake. The decrement in plasma aldosterone with SQ 20881 in 26 hypertensive subjects (15+/-3 ng/dl) was normal (13+/-4 ng/dl), suggesting that the altered adrenal responsiveness in hypertensives is not because of a change in a postreceptor event or in the relative contribution of angiotensin to the control of aldosterone secretion. Saralasin at a dose (0.1 mug/kg per min) that reduced aldosterone levels in all normals produced a normal aldosterone decrement (14+/-3 ng/dl) in 19 patients with renovascular hypertension (12+/-4 ng/dl). The same dose, however, had no net effect on plasma aldosterone levels in 70 patients with normal or high renin essential hypertension (-1+/-1 ng/dl) despite identical metabolic balance and control renin and angiotensin levels. The altered response could be explained by an agonist effect, aldosterone rising in 45 of the essential hypertensives. There were no significant differences between normal and abnormal responders in pre- and postcortisol, -potassium, -renin and -angiotensin concentrations. Angiotensin was infused (0.1-3 ng/kg per min) in 15 patients with normal renin essential hypertension, previously studied with saralasin. A probit transformation defined the dose required to induce a 50% increase in aldosterone (ED50). In the patients in whom aldosterone rose with saralasin, the dose required to induce a 50% increase was significantly greater (P < 0.001) than in those in whom aldosterone fell normally (1.02+/-0.06 [SD] vs. 0.38+/-0.07 ng/kg per min). Vascular responses were similar in the various groups. We conclude that altered adrenal responsiveness to angiotensin in some essential hypertensive patients is secondary to a change in the interaction of angiotensin with its adrenal receptor.


Hepatitis B virus antigens in primary hepatic carcinoma: immunofluorescent techniques on fixed liver tissue. The presence of hepatitis B surface (HBsAg) and core (HBcAg) antigens was investigated by immunofluorescence in specimens of liver tissue obtained at necrospy in 107 patients with primary hepatic carcinoma. HBsAg was detected in the cytoplasm of liver cells in 16 cases, and in eight of them the antigen was also found in malignant cells. HBcAg, which was present in the nuclei of liver cells in eight cases, was detected in the nuclei of tumour cells in six of these and also in two other cases showing HBsAg, but not HBcAg, in the nonneoplastic tissue. Although most of the primary hepatic carcinomas studied were associated with cirrhotic changes in the non-neoplastic tissue, HBsAg and HBcAg were also detected in the absence of underlying cirrhosis. Hepatitis B virus markers were demonstrated in non-neoplastic tissue, mainly in patients with a well-differentiated carcinoma, and only in these cases were they found also in the neoplastic tissue. These results show that hepatitis B virus antigens, including HBcAg, can be detected in the neoplastic cells of well-differentiated carcinoma of the liver. Although these cells could have been infected after the malignant transformation, a direct oncogenic role of the virus cannot be excluded.


Continuous-flow analysis of ammonia in perchloric acid supernate of blood or plasma using an ammonia-selective electrode. A simple automated method for the estimation of ammonia in perchloric acid supernate of blood or plasma using an ion-selective electrode (Orion Ammonia-selective electrode, Model 95-10) is described. The reliability of the proposed method has been checked against an ion-exchange resin procedure, which has been chosen as a standard procedure. Regression equation and correlation coefficient for the proposed method are y = 0.7 X + 10 and 0.945, respectively, as compared with the chosen standard method. Within-run and between-run precision are 2.1% and 3.5% respectively. The average percent recovery is 97.5% and a tentative range is 13-73 microgram/dl (9-52 micronmol/l) ammonia nitrogen.


Mucin-producing atypical bronchial carcinoid. In a case of atypical mucin-producing bronchial carcinoid in a 40-year-old man covert metastases were present at the time of lobectomy and were revealed at necropsy--a feature highlighting the malignant potential of the atypical carcinoid.


Viruses in the stools.

Viruses in the stools. It has long been possible to isolate viruses from the stools by culture, though the viruses found are rarely implicated in disease of the gut. In contrast, only recently has it been possible to identify viruses in the stools of patients with diarrhoea. Initially, such identifications were made by electron microscopy but the unsuitability of the microscope for large-scale screening has led to the development of other methods. The new methods have concentrated on rotaviruses but other viruses are also implicated and an overall view of the significance of finding a virus in any stool specimen has to take into account the evidence about all viruses, old and new.


Granular cell myoblastoma: positive staining for carcinoembryonic antigen. An immunoperoxidase technique for the detection of carcinoembryonic antigen was applied to 10 cases of granular cell myoblastoma. Consistent, strong, intracytoplasmic granular staining, which can be easily interpreted, was obtained in all cases. Schwannomas, neurofibromas, dermatofibromas, and leiomyomas were negative. The test is helpful in confirming doubtful cases. The results tend to support the suggestion that granular cell myoblastoma is derived from perineural rather than endoneural cells.


Detection of inferior vena cava abnormalities by computed tomography. The diagnostically useful computed tomography (CT) findings in five new cases of inferior vena cava thrombosis are reported. These findings include: (a) inhomogeneous density of the inferior vena cava, usually best demonstrated after contrast medium injection; (b) caval density less than aortic density; (c) rim enhancement which may be seen with both caval thrombosis and tumor invasion; and (d) enlargement of the vena cava if accompanied by at least one or more of the above findings. A bolus injection of contrast medium followed immediately by CT helps to make some of the above described findings more prominent. In selected patients, this injection should be made into the femoral vein.


Decreased cAMP levels in human diploid cells exposed to cholinergic stimuli. Carbachol at a concentration of 1 micron caused very significant decreases in PGE1 stimulated cAMP levels in three human diploid lung fibroblasts, WI-38, MRC-5, and IMR-90. Detailed studies with WI-38 cells showed that carbachol acted at low concentrations and very rapidly, cAMP levels being reduced by 50% in about 1 minute. The effect was moderately reduced but still very significant in Ca++ free medium containing 10(-5)M EGTA. Acetylcholine (in the presence of physostigmine), pilocarpine, and muscarine lowered cAMP levels at concentrations of 1 or 10 micron. The effects of carbachol were exquisitely sensitive to atropine but were unaffected by hexomethonium or d-tubocurarine. These data suggested that the cholinergic response of WI-38 cells was of the muscarinic type.


Quantitative labeling of the regulatory subunit of type II cAMP-dependent protein kinase from bovine heart by a photoaffinity analog. Several methods were compared for estimating the amount of regulatory subunit of an 800-fold purified Type II cAMP-dependent protein kinase from bovine heart. These methods included a reversable binding assay using either cAMP, or 8-N3-[32P]cAMP, photoaffinity labeling with 8-N3-[32P]cAMP, and autophosphorylation of the regulatory subunit of the enzyme. Although the regulatory subunit had a slightly lower affinity for 8-N3-cAMP than for cAMP, the total amount of regulatory subunit could be determined by each of the procedures examined. The results indicate that the photoaffinity analog 8-N3-[32P]cAMP is able to label quantitatively all cAMP-binding sites of the regulatory subunit of this cAMP-dependent protein kinase.


Protein kinases in developing rat brain. The levels of cAMP-dependent protein kinases were measured in developing rat brain by a variety of methods. The regulatory subunit (R) was measured both by [3H]cAMP binding and by 8-N3-[32P]cAMP incorporation. The catalytic subunit (C) was measured by an assay of histone kinase activity. Data were calculated per mg protein. Neither R nor C levels changed significantly in either membranes or cytosol during development. The ratio of R to C was essentially unity in the cerebra of both newborn (2-day-old) and adult (40-day-old) rats. Polyacrylamide-gel electrophoresis resolved two regulatory subunits (R-I) and (R-II) which were derived from the Type I and Type II cAMP-dependent protein kinases, respectively. 8-N3-[32P]cAMP incorporation into Proteins R-I and R-II indicated that the amounts of Proteins R-I and R-II did not change significantly in either membranes or cytosol during development.


Actomyosin from mammary myoepithelial cells and phosphorylation by myosin light chain kinase. The oxytocin-sensitive myoepithelial cells of the mammary gland form a system with characteristics of a potentially useful model for studying the mechanism of action of oxytocin and coupling phenomena of excitation-contraction. Our objectives were to develop a method for isolating mammary actomyosin, to determine the amount of actomyosin in the glands of lactating and nonlactating animals, and to investigate control of contractile protein interaction. Actomyosin in mammary glands represented a substantial portion of the soluble protein in the gland ranging from 9% of the total in lactating to 17% in weaned rats. The isolated actomyosin had a molecular composition like that of actomyosin of smooth muscle and the isolated actomyosin contained a light chain kinase that phosphorylated the 20,000 dalton light chain of myosin (L20). The kinase isolated as a component of actomyosin preparations did not show calcium control, but it did when isolated from mammary cytosol. Strips of involuted mammary tissue from rats developed tension when oxytocin was added to the bathing medium; thus, the myoepithelial cells appeared to retain their sensitivity to oxytocin even in nonlactating animals and may be a useful model for studying the action of oxytocin. We suggest that one of the final steps in the milk-ejection reflex is phosphorylation of myosin causing a contraction of the myoepithelial cells of the mammary gland.


Mobilizing nursing skills.

Mobilizing nursing skills. The author argues that the practice of nursing should be based on the "nursing model" related to care not the "medical model" related to cure. Using the nursing model entails making an assessment of the patient's needs and an individualized nursing care plan to meet them. It is conceded that the historical development of nursing practice as a series of stereotyped, ritualized activities within a hierarchical profession militates against implementing the nursing model; but the author illustrates how to Royal Marsden Hospital, a specialist hospital for the treatment of cancer patients, has innovated and developed the concept, as well as creating specialist roles for nurses who maintain continuity of patient care from the first appointment in the hospital's outpatients' clinic through to this after-care following his discharge home.


Changes in hormonal activities relative to the severity of essential hypertension. Endocrine activity in patients with essential hypertension was studied by measuring the urinary excretion of catecholamines, prostaglandin E (PGE) and cyclic adenosine monophosphate (cAMP). Simultaneously, plasma renin activity, concentrations of serum sodium, potassium, blood urea nitrogen (BUN) and creatinine were determined. Systolic blood pressure and BUN increased progressively with age until the sixth decade. Urinary excretion of norepinephrine was correlated with the systolic blood pressure. In contrast, plasma renin activity and urinary excretion of PGE decreased progressively with the increase in systolic blood pressure. Although the cause of essential hypertension is not known, it is suggested that hypertension accelerates the aging process in the kidney and thus decreases renal PGE synthesis. This decrease of PGE in turn causes a reduction of plasma renin activity, possibly either by accelerating the retention of sodium and water or by failing to stimulate renin synthesis. A decrease of PGE may also potentiate the vasopressor action of norepinephrine.


[Ocular herpes : clinical variations and virus antigenic type (author's transl)]. Fifty-seven herpes simplex virus strains, isolated from ocular herpes, were screened by complement fixation, seroneutralization and thermosensitivity tests. They were distributed as follows : 44 type 1, 3 type 2, 7 intermediate type ; 3 had a type 2 thermosensitivity and a type 1 neutralization. Discrepancies in these results, with the three tests, show that there is, in each typed group, strains of virus which react differently, and this could indicate a continuity from one type to another. As the strain progressively reacts more like the type 2, clinical symptoms become more severe. In a certain number of cases where several simultaneous or successive samples were performed, there was a hypothetic possibility of change in the behaviour of herpes simplex virus strains in the eye.


Effect of age and sex on triglyceride metabolism in the rat. The effect of age and sex on triglyceride metabolism were investigated in 3-mo- and 1-year-old rats. Although 3-mo-old female rats produce as much triglyceride as comparably aged male rats, their plasma triglyceride levels are lower. This suggests that 3-mo-old female rats remove triglycerides from plasma more efficiently than do similar aged male rats, and this is supported by the observation that the t1/2 of very low density lipoprotein triglyceride removal is somewhat faster in female rats of this age (p less than 0.1 greater than 0.5). As rats grow to 1 year of age, the ability of both sexes to remove triglycerides from plasma deteriorates, resulting in a rise in plasma triglyceride levels. The decrease in efficiency of triglyceride removal from plasma in female rats is coupled with an increase in triglyceride production rate, leading to an even greater increase in triglyceride levels. These results indicate that both age and sex modify triglyceride metabolism in the rat.


Some age-related changes in pituitary-adrenal function in the male laboratory rat. Changes in the pituitary-adrenal axis have been investigated as a function of age (2 1/2 to 26 mo) in the male laboratory rat (CFY-Sprague Dawley). Ether stress was used to challenge the pituitary-adrenal axis and blood samples (peripheral and adrenal venous effluent) taken for measurement of corticosterone using the competitive protein-binding assay and ACTH by the increase in corticosteroidogenesis in isolated rat adrenal cells. The results show that there was an increase of basal ACTH levels with increase in age which might be correlated with the degenerative changes in the adrenal cortex at old age. No age difference was observed in the 2 1/2-min stress levels. However, 15-min stress resulted in a further increment of ACTH levels, which was not found in old animals. No age-related differences in the basal and stress levels of corticosterone was observed, and, hence, adrenocortical function would seem not to be impaired in old age.


The effect of relative humidity on swine vesicular disease virus in dried films before and during formaldehyde fumigation. Swine Vesicular Disease virus (SVDV) did not survive drying at high relative humidities (r.h.) but there was little virus loss at low r.h. Purified virus dried in films was inactivated by formaldehyde fumigation only at high r.h. Inactivation was also influenced by the suspending medium from which the virus was dried. Purified virus resuspended in distilled water and then dried, was rapidly killed, but that in tissue culture fluid survived.


The inactivation of viruses in cattle and pig slurry by aeration or treatment with calcium hydroxide. Porcine enterovirus type 2 or porcine adenovirus type 3 were seeded into samples of pig slurry, and a bovine enterovirus was seeded into cattle slurry, and samples of the slurry were aerated in the laboratory for 21 days. The viruses were inactivated more rapidly in the aerated slurry than in control slurry which was not aerated. The difference in inactivation rate was greatest for the porcine adenovirus and least for the bovine enterovirus. Inactivation of the porcine enterovirus in aerated distilled water and in aerated, autoclaved pig slurry proceeded at a similar rate as in the same materials which were not aerated. Ten samples of aerated slurry were collected from an aeration tank which received weekly additions of raw pig slurry which was sampled at the same times. Each sample yielded a porcine enterovirus after concentration with the polyelectrolyte PE-60, but in three comparative titrations the viral infectivity titre in concentrates of the raw slurry was at least 1000 times greater than in the aerated slurry. Porcine enterovirus type 2 and porcine adenovirus type 3, which were seeded into pig slurry, and a bovine enterovirus seeded into cattle slurry, were inactivated by treatment of the slurry with calcium hydroxide at pH 11.5. The inactivation rate was highest for the bovine enterovirus and lowest for the porcine adenovirus.


Improved performance of a double antibody radioimmunoassay for carcinoembryonic antigen. A new double antibody solid-phase radioimmunoassay (RIA) for carcinoembryonic antigen (CEA) is critically analyzed. The aim of the study was 4-fold: (a) to define the level of sensitivity (a comparison of 3 different assay procedures revealed that our sequential assay was more sensitive than most previously reported RIAs, while competitive and non-equilibrium assay had wider measuring ranges); (b) to analyze recoveries of CEA in either serum, plasma or urine (the recovery, even in urine, was very close to expected values, indicating that no CEA is lost or degraded during brief storage or in the extraction procedure); (c) to evaluate inter- and intra-assay variations, since most clinical management is dependent on serial assays rather than single determinations. The coefficients of variation were low both within and between assays. A change of 3 ng CEA is required for significant change (greater than 2 S.D.) at the normal serum level which is 16 ng CEA/ml in our assay. At levels above normal, a change of 4 ng is required; (d) the assay was also developed for determination of CEA levels in a large series of perchlorid acid treated serum, plasma or urine samples. This forms the basis for an assay suitable for serial assays with high sensitivity and accuracy in various neoplastic diseases.


Specific immunoglobulin responses after varicella and herpes zoster. The indirect immunofluorescence technique has been used to titrate the specific immunoglobulins in 200 sera from 64 patients with varicella, and 195 sera from 67 patients with herpes zoster. IgG and IgM antibodies were detected in all patients with varicella, and IgA in 59 (92%). All three classes of antibody appeared 2--5 days after the onset of the rash, increased virtually simultaneously and reached maximum titres during the second and third weeks. IgG then declined slowly, but never became undetectable and was still present in five subjects who were retested after 2--4 years; it was present in 88 out of 100 healthy young adults and probably persists indefinitely after varicella. IgA and IgM antibodies declined more rapidly and were not detected in specimens taken more than a year after the illness. IgA, however, may possibly persist in some cases since low titres were found in 8 out of 88 young adults who possessed IgG antibody and had presumably had varicella in the past. IgA responses were significantly weaker in children under the age of 6 years than in older children and adults. Six out of 67 patients with zoster were tested at various times before the onset of the rash: IgG antibody was detected in all. IgG was present in all sera taken after the onset of the rash, increased rapidly after 2--5 days, reached maximum titres during the second and third weeks and then declined slowly. IgA antibody was detected in 66 patients (99%) and IgM in 52 (78%); both types of antibody followed transient courses, as in varicella. Maximum titres of IgG and complement-fixing antibodies were greater after zoster than after varicella, but the differences were not significant. IgA and IgM titres in young adults with zoster were significantly lower than in older patients, and also lower than in young adults with varicella. Increases in varicella-zoster antibody in patients with herpes simplex virus infections consisted mainly of IgG, sometimes IgA, but never IgM.


Neutrophil myeloperoxidase: a simple, reproducible technique to determine activity. Neutrophil MPO deficiency has been rarely documented. This is because of the low incidence of infections in these individuals and the lack of use of various screening techniques which would detect them. Using the Hemalog D, we have identified one completely deficient and 18 partially deficient subjects. These subjects were studied by staining their peripheral blood smears for MPO and grading the intensity of the stain on 100 consecutive neutrophils (MPO score) as well as by chemically quantifying the MPO activity. There is an excellent correlation between MPO score and quantitated MPO activity. The MPO score is a simple semiquantitative method to estimate MPO activity.


Structure activity relationships between catecholamines and the alpha-adrenergic receptor responsible for the aggregation of human platelets by epinephrine. PEA is a potent inhibitor (Ki approximately 13 microM) of human platelet aggregation induced by epinephrine. This led us to perform an SAR study of a congeneric series of compounds in an effort to identify the molecular components of epinephrine critical to its aggregating effect upon human platelets. Phenylethanolamine was similar to PEA in inhibitory potency. However, hydroxylation of the phenyl ring diminished the inhibitory effect (Ki tyramine approximately 87 microM; Ki octopamine approximately 88 microM). Dopamine, the weakest inhibitor (Ki approximately 150 microM), was a partial agonist capable of inducing platelet aggregation in some samples of PRP. The order of potency of catecholamines as aggregating agents was epinephrine greater than norepinephrine greater than Epinine greater than dopamine. Phenylephrine, the prototype alpha-agonist, did not induce aggregation but was a potent inhibitor (Ki approximately 12 microM) of the aggregation induced by epinephrine. Isoproterenol, the prototype beta-agonist, was neither an aggregant nor an inhibitor of epinephrine-induced platelet aggregation. These findings suggest that the binding of epinephrine to the alpha-adrenergic receptor responsible for platelet aggregation is accomplished by the N-methyl amino group whereas intrinsic aggregating activity is a function of the catechol moiety.


Effects of a chemotactic factor, N-formylmethionyl peptide, on adherence, superoxide anion generation, phagocytosis, and microtubule assembly of human polymorphonuclear leukocytes. FMLP promoted microtubule assembly in PMNs at concentrations which were chemotactic for the cells. At higher concentrations than those required for chemotaxis, FMLP enhanced the adherence of PMNs to nylon glass fibers. With colchicine, PMN adherence was inhibited, but upon exposure to FMLP, PMN adherence could be restored. The effect of FMLP on PMN adherence was transitory and was no longer evident by 5 min. At concentrations similar to those employed in the adherence studies. FMLP induced the cells to briefly generate O-2, since ferricytochrome C reduction was no longer evident by 5 min. Pretreatment of the PMNs with cytochalasin B enhanced the release of O-2 by PMNs exposed to FMLP. On the other hand, there was no effect of FMLP on phagocytosis of C3-coated particles. These results suggest that FMLP induces responsive cells to develop a hyperadherent plasma membrane which is largely independent on microtubule control. Since oligopeptides similar of AMLP are formed in bacteria, it is likely that the action of N-formylated peptides is important in regulating the inflammatory response.


The in vivo metabolism of esterified cholesterol in the plasma high-density lipoproteins of rabbits. The in vivo metabolism of esterified cholesterol in plasma HDL has been studied in rabbits injected with preparations of HDL which had been labeled with 3H in the esterified and free cholesterol moieties. These labeled HDL preparations had been isolated either from the serum of donor rabbits which had been previously injected with 3H-mevalonic acid or from rabbit serum which had been incubated in vitro at 37 degrees with 3H-cholesterol. In terms of subsequent in vivo metabolism, there were no significant differences between the in vivo labeled and in vitro labeled HDL preparations. It was also found that the presence of free 3H-cholesterol in the injected HDL had very little effect on the recipient esterified 3H-cholesterol results. The removal of esterified 3H-cholesterol from the recipient HDL fraction was biphasic, with the initial phase largely reflecting a transfer into the plasma VLDL and LDL fractions rather than a removal from the plasma compartment. In fact, the initial rate of removal from HDL was very much influenced by the distribution of esterified cholesterol mass in the different lipoprotein fractions; the greater the proportion in VLDL and LDL, the more rapid was the initial rate of removal from HDL. A transfer of esterified cholesterol from HDL to VLDL and LDL was estimated to be of the order of 150 to 200 mumol/L of plasma per hour, a value much greater than the reported rate of production of esterified cholesterol in rabbit plasma. The implication that such transfers might therefore have been partially reversible was confirmed in in vitro incubations of labeled HDL and unlabeled rabbit serum.


Generation of superoxide radicals by human peripheral neutrophils activated by chemotactic factor. Evidence for the role of calcium. In response to activation by the synthetic chemotactic factor FMLP, human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction. A dose-dependent increase in the amount of superoxide induced by FMLP over the concentration range of 1 X 10(-8) M to 1.6 X 10(-7) M was observed. Examination of the kinetics of the response revealed large amounts of superoxide generated by 1 min of incubation at 37 degrees C at an optimal dose of FMLP and a plateau effect after 5 min of incubation. Divalent cations did not influence the binding of 3H-FMLP to the cell, but superoxide generation by FMLP-activated neutrophils was observed to be dependent on the presence of divalent cations in the medium. In the absence of Mg2+, increasing Ca2+ ion concentration in the medium led to progressive increases in superoxide generation up to 4 mM, after which the response declined slightly. Mg2+, 0.25 to 4 mM, increased FMLP-induced superoxide generation to a much lower extent than did Ca2+. Lanthanum ion, 0.1 to 1 mM, in the presence of 1 mM Ca2+ inhibited the production of superoxide by FMLP 4 X 10(-8 ) M. Over the concentration range 3.3 X 10(-5 M to 3 X 10(-4 M, verapamil, a drug which selectively blocks the calcium channel, caused a dose-dependent inhibition of superoxide production and calcium-45 uptake in response to FMLP. This effect of verapamil could be overcome by increasing the concentration of Ca2+ in the medium. These observations suggest that a calcium influx plays an important role in the superoxide-generating capacity of the neutrophil.


The effect of homologous thrombin and fibrinogen degradation products on fibrinogen synthesis in rabbits. The effect of intravenous infusions of purified homologous FDP and thrombin on fibrinogen synthesis was evaluated in rabbits. De novo fibrinogen production was measured by the rate of incorporation of 75SeM into circulating fibrinogen. After receiving either 100 or 200 NIH U/kg purified homologous thrombin over 1 hr, rabbits demonstrated threefold and fivefold increases in fibrinogen synthesis, respectively. A correlation between the titers of FDP-fdp and the degree of fibrinogen synthesis was evident. Prior administration of epsilon-ACA prevented the accelerated synthesis of fibrinogen induced by thrombin and inhibited the appearance of FDP-fdp in serum. epsilon-ACA did not interfere with normal fibrinogen production. Fibrinogen synthesis was assessed following infusions of FDP prepared by vitro by digestion of rabbit fibrinogen with plasmin and subsequently identified on SDS-polyacrylamide gels. Preparations which contained predominantly stage 2 intermediate (X, Y, D, and E) or stage 3 final (D and E) fragments accelerated fibrinogen synthesis, whereas those containing predominantly stage 1 fragment X did not. Prior treatment with epsilon-ACA did not alter these results. Infusion of the supernatants derived from immunoprecipitation of the FDP by either anti-rabbit fibrinogen antibody or specific anti-human D and E antibodies significantly diminished the enhanced fibrinogen synthesis induced by the unadsorbed materials. These experiments suggest that the accelerated fibrinogen synthesis induced by thrombin is mediated by FDP, with fragments D and E appearing to be the most potent.


Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity. Conventional methods for determination of membrane-associated Mg- and NaK-ATPase activity typically involve a timed incubation of enzyme with ATP followed by measurement of released Pi. They are time-consuming, require a large amount of enzyme protein, and show considerable variability induplicate samples. These problems can be largely eliminated with a coupled enzyme assay in which formation of ADP by ATPase is coupled to NADH oxidation with the intermediate enzymes PK and LDH and the intermediate substrate phosphoenolpyruvate present in excess. NADH oxidation is continuously recorded at 340 nm, and NaK-ATPase represents the ouabain suppressible fraction of total ATPase. This method allows accurate determinatin of initial reaction rates which vary linearly with respect to enzyme protein. Furthermore, it eliminates the need to measure Pi and prevents accumulation of inhibitory ADP. In over 100 preparations of LPM prepared from rats pretreated with agents known to alter ATPase activity or exposed in vitro to ATPase inhibitors. ATPase activity by the coupled-enzyme assay paralleled results obtained with the conventional method. Moreover, the coupled-enzyme assay required less membrane protein, showed less variability in duplicate samples, required half the time, and also yielded accurate values in brain, kidney, and heart tissue. This improved assay should find broad application in the study of membrane ATPase as they relate to a variety of cellular functions.


Influence of cationic local anesthetics on the metabolism and ultrastructure of human alveolar macrophages. The concentrations of cationic local anesthetics present in effluents from subsegmental bronchoscopic lavage were determined. Subsequently, the effect of these agents on lavaged human AM was evaluated in vitro. The results indicate that concentrations of LDC that may alter human AM function are present in effluents during routine subsegmental bronchopulmonary lavage. LDC and TRC in a dose-dependent fashion rapidly inhibited oxygen consumption and superoxide anion (O-2.) release by unstimulated human AM or AM stimulated by bacteria or the membrane-active chemical PMA. Concentrations of 2 mM TRC or 16 mM LDC reduced O2 consumption and O-2. release by unstimulated AM by more than 70% and blocked the usual spurt in O2 uptake and O-2. release observed for stimulated AM. This inhibition was not due to cytotoxicity, since washing n a balanced salt solution restored the metabolic function of treated AM. TRC or LDC also had effects on the morphology of washed human AM, causing rounding of the cell surface (scanning electron microscopy). In summary, the findings show that anesthetic agents routinely present in lavage effluents have the capacity to alter the function and structure of human AM. Although the effect must be considered in the design and interpretation of studies using AM obtained by bronchopulmonary lavage, the cationic anesthetics may also prove to be valuable agents for evaluating cell membrane-related events of human AM.


Inhibition of aldosterone production in adrenal cell suspensions by serum from patients with manic-depressive psychosis. A method of preparing a suspension of cells of the zona glomerulosa from rat adrenal capsules treated with crude collagenase is described. The cells responded to ACTH, angiotensin II and serotonin by increased production of aldosterone. Pooled human sera or individual human sera (from healthy normal or non-psychiatric in-patients) to a final concentration of 30% had no effect on ACTH-stimulated production of aldosterone. Many serum samples from five patients with manic-depressive psychosis, however, caused a reduction in aldosterone production; 65% of those samples taken during depression, 44% of the samples taken during manic episodes and 23% of the samples taken when the mood was normal. Sera from manic-depressive patients also reduced the production of aldosterone caused by angiotensin II or serotonin. This effect of serum from manic-depressives in vitro may be related to the abnormalities of aldosterone control in such patients.


Comparison of the stimulatory effects of ovine, porcine and human follicle-stimulating hormone and of ovine and human luteinizing hormone on the accumulation of cyclic AMP by porcine granulosa cells. The effects of ovine, porcine and human FSH, and ovine and human LH on the accumulation of cyclic AMP by porcine granulosa cells obtained from follicles at various stages of maturation were investigated. During incubation periods of 15 min, 10 micrograms ovine FSH pretreated with antiserum to LH or 10 micrograms human FSH resulted in an 11- to 18-fold, five- to ninefold, and less than a twofold increase in intracellular accumulation of cyclic AMP by granulosa cells from small (1--2 mm), medium (3--5 mm) and large (6--12 mm) follicles respectively. Similar patterns of response occurred with addition of porcine FSH. After incubation for 30 and 60 min with ovine, porcine or human FSH, significant accumulation of cyclic AMP in the incubation medium occurred with cells obtained from small and medium-sized follicles. After 60 min of incubation with FSH the accumulation of cyclic AMP in the incubation medium exceeded the intracellular cyclic AMP levels in granulosa cells from small and medium-sized follicles. During incubation periods of 15 min, 1.0 micrograms ovine LH resulted in less than a twofold, a fourfold and greater than a tenfold increase in intracellular accumulation of cyclic AMP by granulosa cells from small, medium and large follicles respectively. Addition of human LH brought about a similar response. Incubation periods of 30 and 60 min with 1.0 micrograms ovine or human LH resulted in significant accumulation of cyclic AMP in the incubation medium by granulosa cells from large follicles; cyclic AMP content in the incubation medium was greater after 60 min compared with 30 min of incubation. It was concluded that ovine FSH pretreated with an antiserum to LH had similar effects on cyclic AMP levels as did purified human and porcine FSH, and that the stimulatory effects of the less pure ovine FSH were probably not due to an impurity in the FSH preparation. Porcine granulosa cells obtained from small follicles should be suitable as an in-vitro FSH bioassay while granulosa cells obtained from large follicles should be suitable as an in-vitro LH bioassay.


Regulation of specific cell-mediated cytotoxic response against SV40-induced tumor associated antigens by depletion of suppressor T cells with cyclophosphamide in mice. When cyclophosphamide was administered to mice before immunization with syngeneic SV40 transformed cells, the specific immune response elicited, as was measured by in vitro 51Cr release assay was stronger and lasted longer when compared to the response generated in noncyclophosphamide-treated mice. The augmentation effect of the drug was dependent on cyclophosphamide concentration being optimal at 100 mg/kg and on the time of drug administration in relation to antigen immunization being optimal at 2 d before antigen administration. Transfer of T cells from normal syngeneic mice to drug-treated animals abolished the cyclophosphamide-induced augmentation of immune response. These results implied that cyclophosphamide sensitive T cells suppressed the in vivo generation of specific effector T cells against SV40-induced tumor-associated antigens.


A role for elevated H-2 antigen expression in resistance to neoplasia caused by radiation-induced leukemia virus. Enhancement of effective tumor surveillance by killer lymphocytes. Resistance to neoplasia caused by radiation-induced leukemia virus (RadLV) is mediated by gene(s) in the H-2D region of the major histocompatibility complex. The previous observation that rapid increases in cellular synthesis and cell-surface expression of H-2 antigens are detectable immediately after virus inoculation has suggested that altered expression of H-2 antigens may play a significant role in the mechanism(s) of host defense to virus infection. This concept is supported by the following observations. First, cell-mediated immunity against RadLV transformed or infected cells can be detected with ease when H-2-positive target cells are used in the cell-mediated lympholysis (CML) assay. (Although RadLV transformed cells obtained from overtly leukemic animals and maintained in tissue culture are H-2 negative, these cells can regain their H-2 phenotype by in vivo passage in normal animals. The H-2-negative cells are poor targets in a CML assay.) Second, resistant mice develop greater numbers of effectors when infected with RadLV than do susceptible mice. Third, injection of normal (uninfected) thymocytes into syngeneic recipients of resistant or susceptible H-2 type does not stimulate a CML response. However, injection of RadLV infected thymocytes from resistant mice produces a vigorous CMI response, and such thymocytes elicit the strongest response at a time when both H-2 and viral antigen expression is elevated. By contrast, injection of infected thymocytes from susceptible mice, which express viral antigens, but low levels of H-2 antigens, does not stimulate a CML reaction. These findings may explain the easier induction of leukemia found by many investigators when virus is inoculated into neonatal mice and the preferential thymus tropism of some oncogenic type-C RNA virus. Cells expressing very low levels of H-2, such as thymocytes, may serve as permissive targets for virus infection because they lack an important component (H-2 antigens) of the dual or altered recognition signal required to trigger a defensive host immune response.


Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide. The HL-60 human promyelocytic leukemia cell line can be induced to terminally differentiate to mature myeloid cells sharing a number of functional characteristics with normal granulocytes including response to chemoattractants, development of complement receptors, phagocytosis, superoxide production, and nitroblue tetrazolium dye reduction. Hence the Me2SO-induced HL-60 cells provide a unique in vitro model for studying various important aspects of human myeloid cell differentiation.


The family and cardiac rehabilitation.

The family and cardiac rehabilitation. Family resources and needs are often overlooked in planning for rehabilitation of the individual following myocardial infarction. The acute ischemic event presents a massive disruption in the psychodynamic balance of the family, but it can provide an unparalleled opportunity for increased awareness and growth for all members of the family. An integrated program of emotional support, education, and physical activity is recommended to facilitate restoration of the individual's self-esteem. Formal family counseling provides a forum for resolution of fears and misconceptions and permits redefinition of roles within the family.


Relationship between virus neutralization and serum protection bioassays for IgG and IgM antibodies to foot-and-mouth disease virus. The time interval between administering the serum and the virus was found to influence the results of the in vivo mouse protection test for foot-and-mouth disease antibodies. In particular, for both IgG and IgM antibodies to strain A12 virus, the mouse protection index increased from zero to a maximum at about 6 h and remained high for at least five days. Variations in the antiserum concentration, on a log scale, had a proportional effect on the mouse protection index, if between 1 and 3. The constant of proportionality was unity for IgM and 2 for IgG antibody. Comparison with in vitro neutralization tests revealed essentially parallel neutralization curves. The lower serum titre in the protection test, if computed for less than 10(3) LD50/dose, was accounted for by the simple dilution of the inoculated serum into the volume of the mouse. Consequently, in the low titre range, the same virus-antibody reaction and its effect are operable in each of the two tests. Analysis of literature data in which both the in vivo protection test and the in vitro neutralization test results were available on the same sera showed consistency with the above conclusions for both cattle and swine sera. The protection test had a highly atypical survival pattern occurring at antibody concentrations expected to neutralize more than 10(3) LD50/dose. The resulting in vivo dampening effect on virus titre is postulated to be caused by the excess antibody of the passive immunity test interfering with the spread of infection. The effect is analogous to an anomaly caused by not removing the inoculum in quantal tissue culture assays and it prevents quantification of antibody levels in strong sera.


Isolation and characterization of BK virus-transformed rat and mouse cells. The isolation and characterization of four groups of BK virus (BKV)-transformed rat embryo fibroblast (RE) and mouse kidney (MK) cells are described. They consist of (1) seven RE lines transformed with a BKV pool containing a high proportion of defective virions, and (2) 16 RE, (3) 14 Balb/c-MK and (4) 2 Swiss ICR-MK lines, all transformed, at different input multiplicities, with a pool of BKV free of defective virions. None of the lines produces BKV, all contain BKV T antigen and all grow to higher saturation densities and have higher plating efficiencies than do the corresponding control cells. Cells of the RE lines, transformed with the BKV pool containing defective virions, form colonies in soft agar and produce tumours in irradiated weanling rats, while those of the RE lines transformed with the defective virion-free pool do neither. Cells of the Balb/c-MK, but not of the ICR-MK lines are tumorigenic, although cells of both groups form colonies in soft agar. In general, those lines transformed at higher multiplicities express the biological properties associated with transformation more strongly than do those transformed at lower multiplicities.


Detection and quantification of foot and mouth disease virus by enzyme labelled immunosorbent assay techniques. Enzyme labelled immunosorbent assays (ELISA) have been developed to detect and quantify foot and mouth disease (FMD) virus using flexible plastic microtitre plates. The methods were successful for the specific detection of FMD virus and were 50 to 100 times more sensitive than the complement fixation test. The application of the ELISA techniques to FMD virus typing and subtyping, and to the assay of antigen concentration during manufacture of vaccines is discussed.


Virus carrier state suppresses tumorigenicity of tumor cells in athymic (nude) mice. Nude mice injected subcutaneously with normal uninfected BHK 21 cells or HeLa cells regularly develop large, rapidly-growing tumours at the subcutaneous site of inoculation. However, these same tumour cell lines when persistently infected with VSV or other enveloped RNA viruses are either rejected or form small nodules in nude mice. This rejection phenomenon probably involves some type of immunocyte since heavily-irradiated nude mice (500 rads) cannot reject persistently infected cells but develop large, rapidly-growing tumours which shed virus and defective interfering virus (DI) and which do not exhibit the lymphocytic infiltration observed in the nodules of unirradiated mice given persistently infected cells. Finally, it was possible to select a subline of BHK 21-VSV carrier cells which regularly produces large rapidly-growing tumours in normal unirradiated nude mice, although all these carrier cells express virus antigen and shed large amounts of mature infectious virus and DI both in vivo and in vitro.


Impairment of hormone dependent signal transfer by chronic SSPE virus infection. In a CNS-derived cell line, the cellular response to hormonal stimulation, represented by the rise of intracellular cAMP levels, is impaired under the influence of a persisting neurotropic virus infection. This dysfunction is caused by the decrease in adenylate cyclase activity, most probably due to the virus-induced loss of active catalytic units.


Vertical transmission of murine cytomegalovirus. Congenital abnormalities induced by murine cytomegalovirus have previously been suggested to be an indirect effect of maternal illness as no infectious virus has been isolated from the foetus. However, in this article we report that latent virus detectable by immunofluorescence and in situ hybridization, may frequently be present in foetal tissues.


Rabies virus infection selectively impairs membrane receptor functions in neuronal model cells. A persistent infection with rabies virus (HEP-Flury) was established in the CNS-derived hybrid cell line 108CC15 which possesses specific membrane receptors for prostaglandins, catecholamines and acetylcholine. We report a differential virus influence on the specific receptor response to PGE, isoproterenol and acetycholine as indicated by typical changes of the intracellular cyclic AMP levels. As the adenylate cyclase activity was unchanged in infected cells in vitro, a selective virus influence on specific receptors themselves or their coupling to the cAMP synthesizing system must be considered.


Fine structure of contrasting choroid plexus lesions caused by tertiary amines or cyclophosphamide. Hydropic alterations of the fine structure of the choroid plexus epithelium following administration of three different tertiary amines to rats were compared with alterations produced by cyclophosphamide and with normal choroid plexus. Single doses of 2,7-bis(2-diethylamino)-ethoxy)-4-methyl-1,8-naphthyridine HCl or 2,6-di-(omega-dimethyl aminoethoxy)-pyridine produced dramatic accumulations of membrane-limited vacuoles. Multiple doses of 2,7-bis-(diethylaminoethoxy)fluoren-9-one HCl (tilorone) produced accumulations of dense cytoplasmic bodies in choroid epithelial cells in addition to the hydropic vacuoles. Differential responses to these agents suggested functional specialization or temporal variation in function among adjacent choroidal cells. Despite the occurrence of pronounced plasma exudation in the plexitis following cyclophosphamide treatment, there was no hydropic vacuolization.