Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. IX. Specific inhibition of DNA synthesis initiation by very low density lipoprotein and possible significance to the problem of liver regeneration. Rat serum very low density lipoprotein (VLDL) inhibits initiation of DNA synthesis in fetal rat hepatocyte cultures; cells engaged in synthesizing DNA resist inhibition. VLDL action is specific and apparently blocks prereplicative protein synthesis. These and other results, from studies of altered blood VLDL levels and [3H] thymidine incorporation into isolated liver nuclei in 70% hepatectomized normal and mutant hyperlipoproteinemic rats, as well as from infusion studies with a "mitogenic" hormone solution, suggest that hepatic VLDL metabolism is linked to the suppression of hepatocyte proliferation.


An analysis of lectin-initiated cell agglutination in a series of CHO subclones which respond morphologically to growth in dibutyryl cyclic AMP. We have investigated the molecular basis of the agglutinability of CHO subclones which respond differentially in terms of morphology and surface architecture in the presence of dB-cAMP in the medium. We have demonstrated that the agglutinability of these subclones with both wheat germ agglutinin (WGA) and concanavalin A (Con A) probably depends on the free lateral mobility of the lectin receptor sites in the plane of the membrane. The nonagglutinable surface architecture seems to depend on the presence in the membrane of a protease-labile peptide(s), which appears to be distinct from the lectin receptors, as well as on continuous protein and RNA synthesis. This dependence on continuous transcription  and translation may be related to the maintenance of the protease-labile peptide(s) in such a state as to restrict mobility of the lectin receptors. The surface architecture defined as nonagglutinable also depends on the state of polymerization of the intracellular microtubules and microfilaments. It is suggested that these microskeletal elements serve to anchor the lectin receptors in such a manner as to restrict their mobility and thereby reduce the relative agglutinability of a cell line. We suggest that control of the free mobility of both the Con A and WGA receptor sites is dependent on two constraints, one applied by protease-labile ("surface") membrane components and the other by components of the intracellular microskeletal system.


Metabolism of 125I-labeled lipoproteins by the isolated rat lung. The capacity of the isolated perfused rat lung to metabolize the protein moieties of serum lipoproteins was assessed using homologous (rat) and heterologous (human) plasma lipoproteins. The protein and lipid moieties of the plasma lipoproteins were labeled in vivo with Na[125I]. In selected cases the lipoprotein peptides were labeled in vivo with 14C- or 3H-labeled amino acids. Uptake of lipoprotein label during perfusion was monitored by measure of losses in perfusate label and by rises in pulmonary tissue labeling as shown by radioassay and by light and electron microscope radioautography. Lipoprotein degradation was assessed by fractionation of perfusate and lung tissue radioactive material into trichloroacetic acid (TCA)-isoluble, TCA-soluble, and ether-ethanol-soluble fractions. When heparin was included in the perfusion medium, there was selective degradation of the protein portion of very low density lipoprotein (VLDL) in the perfusate and concomitant uptake of radioactive label by the lungs. Low density lipoprotein (LDL)) was neither taken up nor catabolized by the isolated rat lung in the absence or presence of heparin. By light and electron microscopy, the label was localized over the interalveolar septa, predominantly the capillary endothelium. Disappearance of TCA-insoluble radioactivity from the perfusate was associated with the generation of both TCA-soluble iodide and noniodide radioactivity. Greater than 50% of the radioactive label taken up by the lungs was found in the delipidated TCA-insoluble fraction. This study provides in vitro evidence for pulmonary catabolism of VLDL apolipoproteins and uptake of peptide catabolic products of VLDL by the lung.


Regular structures in unit membranes. II. Morphological and biochemical characterization of two water-soluble membrane proteins isolated from the suckling rat ileum. Specialized plasma membranes from the endocytic complex of ileal epithelial cells of suckling rats were isolated by differential flotation. Thin-section and negative-stain electron microscopy showed the luminal surfaces of these membranes to be covered by an ordered array of particles 14.5-nm separations in long rows. This particulate coating was released from the membrane surfaces by 10 mM CaCl2 and recovered free of membranes after dialysis against 0.5 mM EGTA and high-speed centrifugation. Two proteins were resolved by gel filtration to be in supernate: n-acetyl-beta-glucosaminidase and a filamentous protein which attaches n-acetylglucosaminidase to the membrane surface thereby providing bidirectionality to the array of enzyme. We believe that the filamentous protein has not been previously described. Therefore we have called it ligatin from the latin ligare, which translates "to bind together". Furthermore, we suggest that the membranes of the endocytic complex contain sites for the extracellular digestion of carbohydrate moieties in the maternal milk.


BHK21 fibroblast aggregation inhibited by glycopeptides from the cell surface. Glycopeptides were removed by trypsinization from the surface of baby hamster kidney cells (line BHK21-C13), digested by pronase and separated into 2 fractions by exclusion chromatography. The addition of small amounts of either glycopeptide fraction to shaken suspensions of lightly trypsinzied cells inhibited their rapid aggregation, but one fraction was more active than the other and in higher concentrations it was able to inhibit aggregation completely. After this fraction was purified by high-voltage electrophoresis one subfraction also inhibited aggregation. The effect of the glycopeptides increased following their pretreatment with neuraminidase, but preincubation with periodiate or galactose oxidase destroyed all activity. Galactose oxidase also inhibited cell aggregation directly. Similar glycopeptides from virus-transformed BHK21 cells, oligosaccharides and intact and desialysed human urinary glycoproteins had comparatively little or no effect on BHK21 cell aggregation. The results suggest terminal beta-galactosides and possible alpha-galactosides, and to some extent a particular substructure of cell surface heteroglycans are necessary for their inhibitory activity. The parent, plasma membrane of glycoproteins might serve as adhesive binding sites in cell cohesion, but some evidence indicates cell surface sialyl- and galactosyltransferases may not ordinarily act as their complementary binding receptors.


[Masson's tumor of the fingers].

[Masson's tumor of the fingers]. The authors report three cases of glomus tumour of the fingers and conclude, from the angiographic appearance, that the glomus tumour presents direct and indirect signs suggesting the lesion.


[Quantitative separation of biological lipids by successive chromatographic runs on juxtaposed thin layers (author's transl)]. During a study on incorporation of 14C-labeled precursors in biological lipids, we had to elaborate a method of quantitative separation of lipid classes, based upon three successive chromatographic runs on juxtaposed thin layers. The first thin layer, on which the lipid extract is applied, consists of silica gel alkalised by means of sodium hydroxide solution. The second thin layer is constituted only by silica gel G. The three one-dimensional migrations are respectively carried out in the following solvent systems: diethyl ether-hexane-benzene (55:30:15), hexane-benzene (80:20) and diethyl ether-benzene-acetic acid (62:20:18). The lipid fractions are scraped out and counted by liquid scintillation. The reproducibility of the whole operation is discussed.


[Lysergic acids. II. Isolation and separation of lysergic acids (author's transl)]. During studies on the isolation of both lysergic acids, D-and D-iso-, from hydrolytic mixtures of ergot alkaloids, it became necessary to find a simple chromatographic system for column isolation of lysergic acids. The column with controlled pore glass is highly effective for these purposes. The separation of both isomeric lysergic acids occurs on the column with silica gel. The assay and the composition of lysergic acid are estimated by thin-layer chromatography on precoated plates.


Forensic aspects of high-pressure liquid chromatography. This paper reviews the applications of high-pressure liquid chromatography (HPLC) to forensic problems, and discusses some of the developments that have taken place in the use of the technique in the Metropolitan Police Laboratory. Preparation of octadecyltrichlorosilane-modified silica is described and some of the chromatographic characteristics of this material are investigated. Applications of HPLC to the analysis of cannabis, opium alkaloids, amphetamine-related materials, LSD and polynuclear hydrocarbons are described.


Dehydroepiandrosterone sulfate (DS) levels, a rapid test for abnormal adrenal androgen secretion. Dehydroepiandrosterone sulfate (DS) concentration was measured in the sera of premature and full-term infants and in children throughout puberty. Panhypopituitary, Addisonian, and virilized children were also studied. DS decreased slowly during the first weeks of life from a high level in neonates to the low levels observed between one to five years. After five years of age, DS concentration started to rise. A steeper increase was observed with the onset of puberty, and adult DS concentrations were reached in late puberty. There was no sex difference in DS concentration at any pubertal stage or bone age. Day-to-day variations were small in childhood and during puberty, but were considerable in premature infants. DS concentrations measured at 0900 h were not significantly different from those at 1700 h. There was a positive correlation of serum DS concentrations with the excretion of urinary 17-ketosteroids in boys and girls (r=0.789). Premature infants had DS concentration in or above the late pubertal range. Five panhypopituitary patients and five Addisonian patients had DS concentrations below normal. DS was markedly elevated in patients with congenital adrenal hyperplasia and in one girl with adrenal carcinoma, and was suppressible with dexamethasone in the former. The ease of measurement and the small amount of blood required make serum DS determination a useful guide for adrenal androgen secretion.


Testosterone-estradiol-binding globulin in patients with Turner's syndrome: effects of estrogens and acute growth hormone administration. The TeBG activity of plasma from patients with Turner's syndrome was measured quantitatively using polyacrylamide gel electrophoresis. Human growth hormone administration did not significantly change the plasma TeBG levels. However, oral replacement therapy with estrogens elevated plasma TeBG within 3 days; after 9 days these levels reached a maximum of three- to four-fold greater than that observed at a time prior to therapy.


Evidence for altered synthesis of human chorionic gonadotropin in gestational trophoblastic tumors. Tissue extracts of molar tissue or choriocarcinoma, blood, or urine of 21 women with gestational trophoblastic disease were analyzed for hCG and its subunits. The extracts or biologic fluids were initially chromatographed through a standardized Sephadex G-100 column. Each fraction was radioimmunoassayed in homologous hCG, hCGalpha, and hCGbeta assays. Extracts of four hydatidiform moles contained primarily hCG but no free alpha subunit of that hormone. One of the molar extracts contained a small amount of free hCGbeta not observed in the small amount of free hCGbeta not observed in the extracts of the other three moles. Plasma and urine samples from 18 women with localized or metastatic gestational trophoblastic disease contained hCG but no free alpha or beta subunits; the neoplasms of those patients readily responded to chemotherapy, and all patients have had no evidence of disease for at least one year. The major portion of immunologic hCG present in the biologic samples was indistinguishable from native hCG in its physical behavior on Sephadex G-100 chromatography. On the other hand, three women with widely metastatic tumors died in spite of extensive chemotherapy. Extracts of tumors from two of those patients contained hCG and free subunits of hCG. The urine and plasma of the third patient contained hCG and hCGalpha with the concentration of hCGalpha far exceeding that for hCG in urine. The results of these studies clearly show a disparity between the forms of hCG found in the normal placenta and pregnancy sera, as previously reported, and those found in the neoplastic trophoblast with varying degrees of anaplasia. The most striking finding was the absence of free circulating hCGalpha in the sera in patients with gestational trophoblastic disease which responded to chemotherapy, since free hCGalpha is readily detectable in the sera of normally pregnant women.


Effects of ACTH on steroid metabolism.

Effects of ACTH on steroid metabolism. Metabolic clearance rates (MCR) of aldosterone, cortisol, DOC, corticosterone, and progesterone were simultaneously measured by constant infusion in 8 control subjects before and during an ACTH infusion (12.5 U/4 h). Plasma levels of aldosterone and cortisol, the heat-labile plasma protein-bound fraction of aldosterone, and hepatic blood flow as estimated by the fractional clearance of indocyanine green, were concomitantly determined. The mean MCR of aldosterone, cortisol, and DOC increased to a peak value of 26%, 23%, and 22%, respectively, above baseline values. The increase was already significant (P is less than 0.001) 30 min after the beginning of the ACTH infusion. In contrast, no consistent changes occurred in the MCR of corticosterone and progesterone or in hepatic blood flow. ACTH induced a marked fall in the heat-labile plasma protein-bound fraction of aldosterone. Plasma levels of aldosterone and corsitol increased by 190 and 250%, respectively. A significant negative correlation (r=-0.423, P is less than 0.01) between the MCR and the heat-labile protein-bound fraction of aldosterone was observed. We suggest that competitive interactions in the protein binding properties of various steroids account for the selective effect of ACTH on the MCR of aldosterone, cortisol, and DOC.


Observations on the cyclic nucleotide concentrations in human cerebrospinal fluid. Previous studies have shown that the concentrations of 3', 5' cyclic adenosine monophosphate (cAMP) and 3', 5' cyclic guanosine monophosphate (cGMP) in cerebrospinal fluid (CSF), brain, or both, are increased by melanotropic peptides and catechol amines, and by cholinergic agents. The present study measured the concentrations of cAMP, cGMP, and melanotropic activity in the CSF of normal patients and in 136 subjects with various neurologic diseases. In normal lumbar CSF, concentrations (ave +/- SD) were: cAMP, 21 +/- 8 mM; cGMP, 2.4 +/- 0.5 mM; melanotropic activity, 17 +/- 6 units/100 ml. Concentrations of cAMP, cGMP, and melanotropic activity did not differ significantly (P is less than .05) from normal in the following categories of adult and pediatric patients: back pain due to vertigo of unknown cause; cerebral atrophy; cerebral vascular disease; and brain tumor subdural hematoma not causing increased ventricular pressure. Nine children with retarded psychomotor development caused by diffuse brain disease (infection, trauma, hemorrhage, degenerative process, long-standing hydrocephalus with thinning of the cerebral mantle) had subnormal levels of cAMP and melanotropic activity. These two variables were significantly correlated in the entire series of CSF samples (r=+0.55, P is less than .005). cGMP was elevated in the ventricular fluid of adult and pediatric patients when the ventricular pressure was abnormally elevated. The nucleotide's level rose as high as 50 X normal when ventricular pressure exceeded 300 mm H2O. The concentration of ventricular cGMP was proportional to that of ventricular pressure (r=+0.76, P is less than .005). The correlation was similar regardless of the type of hydrocephalus (congenital or acquired, communicating or obstructive), the age of the patient, or the nature of the underlying disease.


Method for preparation of ampoules containing microgram quantities of polypeptide hormones for distribution and use as laboratory standards: synthetic human ACTH and beta-MSH. There are a number of polypeptide hormones, their subunits, and other derivatives that are available in such short supply as to preclude distribution by conventional methods to the many qualified investigators who require them. This report describes a method for preparing sealed ampoules of two such hormones, the utilization of which can provide many qualified investigators with comparable, stable reference compounds for several years and eliminate unnecessary waste and error associated with the distribution and use of bulk quantitites of peptide hormone preparations. Sealed ampoules of synthetic human adrenocorticotrophic hormone (alpha(h)(1-39) ACTH) and beta melanocyte-stimulating hormone (beta(h)(1-22) MSH) were prepared. Each ampuole contains 50 mug of dried and recoverable peptide plus 1 mg of lactose, sealed under dry nitrogen at atmospheric pressure. The respective preparations are fully active, both biologically and radioimmunologically, and are suitable as reference compounds in all types of biologic and radioimmunologic assays of these hormones. In addition, radioactive iodine labeling of each peptide can be accomplished without prior removal of the lactose.


Laboratory investigation of diarrhea in travelers to Mexico: evaluation of methods for detecting enterotoxigenic Echerichia coli. A laboratory investigation was conducted on cultures collected from travelers before, during, and after a trip to Mexico to characterize the etiology of traveler's diarrhea. Four laboratory methods for detecting enterotoxigenicity of Escherichia coli were evaluated: the infant mouse assay, the Chinese hamster ovary (CHO) cell assay, the Y1 adrenal cell assay, and the rabbit ileal loop. Although a number of common enteric pathogens were identified as a cause of traveler's diarrhea, including six serotypes of Salmonella, two serotypes of Shigella, Vibrio parahaemolyticus, Giardia lamblia, and Entamoeba histolytica, enterotoxigenic Escherichia coli was most commonly isolated. Strains were identified that produced only heat-labile enterotoxin (LT), only heat-stable enterotoxin (ST), or both LT and ST. The infant mouse assay yielded results falling into two distinct groups, providing a clear separation of positive and negative cultures. The CHO assay also formed two groups, with positive cultures producing 11% or more of the elongated cells. There was good agreement between the CHO and the Y1 adrenal cell assays for detection of LT. The adrenal cell system for detection of LT was more suitable than the CHO assay for processing large numbers of specimens because of the miniculture modification of this method utilized in this study. The infant mouse method was a simple and reliable method for detecting ST.


Comparison of rhinovirus-sensitive HeLa cells and human embryo fibroblasts for isolation of rhinoviruses from patients with respiratory disease. Rhinovirus-sensitive HeLa cells (HeLa "R") and human embryo lung fibroblasts (HEL) were compared for the isolation of rhinoviruses from patients with respiratory disease. In the period May 1970 to December 1974, 526 rhinoviruses were isolated from 517 patients, 32% in both cell types, 59% in HeLa "R" only, and 9% in HEL only. The annual isolations in HeLa "R" were between 1.7 and 4 times greater than in HEL cells and may have been due to changes in the sensitivity of the cells or the prevalence of serotypes favoring the HeLa "R" cells. Acid lability was more easily demonstrated in HeLa "R" than in HEL cells, because the infectivity titers obtained were 10- to 100-fold higher.


Application of a new method for detecting streptococcal nicotinamide adenine dinucleotide glycohydrolase to various M types of Streptoccus pyogenes. The production of extracellular nicotinamide adenine dinucleotide glycohydrolase (NADase) by Streptococcus pyogenes can easily be demonstrated using the fluorescence of nicotinamide adenine dinucleotide in ultraviolet light occurring on addition of strong alkali. The new method described here uses microtiter plates and can be read with the naked eye. It permits the screening of large numbers of strains in a short time and with minimal amounts of reagents. The sensitivity of the new method proved to be good in comparison with the bisulfite spectrophotometric method. Culture supernatants of 177 group A streptococci were tested for NADase production by the microtiter fluorescence method. We could confirm former findings that strains within a certain M type of S. pyogenes are usually either producers or nonproducers of the enzyme. The usefulness of the test for screening of streptococcal NADase production is discussed.


Suppression by cyproheptadine of human growth hormone and cortisol secretion during sleep. The effect of cyproheptadine on plasma growth hormone and cortisol levels was studied in seven male volunteers with polygraphic sleep monitoring. Sleep-related growth hormone release was completely inhibited in three of the seven normal subjects by the intravenous infusion of cyproheptadine (5 mg) which was started at the onset of sleep. In the other four, growth hormone release during sleep was significantly decreased or delayed by cyproheptadine when the drug infusion was started at 7:00 p.m., 1-2 h before the onset of sleep. The usual increase in plasma cortisol in the early morning was completely suppressed in all five subjects given cyproheptadine infusions from 4:00 to 7:00 a.m. The intravenous infusion of cyproheptadine increased slow wave sleep, although the time from sleep onset to the first occurrence of slow wave sleep was not affected. In contrast, rapid eye movement sleep was significantly decreased by cyproheptadine. These results suggest that cyproheptadine inhibits growth hormone and ACTH secretion during sleep in man, possibly by antagonizing serotoninergic mechanisms although other actions of the drug are not ruled out.


Hormonal modulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity in rat renal cortex. Specificity of enzyme translocation. Many of the intracellular actions of cyclic adenosine 3',5'-monophosphate are expressed through phosphorylation reactions mediated by cAMP-dependent protein kinases, but little is known about hormonal control of endogenous protein kinase activity (PK) in kidney. In the present study, we examined the effects of parathyroid hormone, glucagon, and isoproterenol on cAMP and PK in slices of rat renal cortex. In the presence of 0.5 mM 1-methyl, 3-isobutyl xanthine, all three hormones activated PK in slices, as reflected by an increase in the ratio of enzyme activity assayable in homogenates of the slices without addition of cAMP to the kinase reaction mixture (cAMP-independent activity) over total enzyme activity (+2 uM cAMP in the reaction mixture). When enzyme activity was assayed in whole homogenates prepared from slices, the increase in the enzyme activity ratio (- cAMP/+cAMP) which followed hormonal stimulation was due entirely to an increase in cAMP-independent activity, with no change in total activity. In general, a good correlation existed between the alterations in tissue cAMP levels mediated by the hormones and/or 1-methyl, 3-isobutyl xanthine and concomitant alterations in PK. All three hormones increased PK activity ratios to near unity, suggesting complete enzyme activation. However, the concentrations of parathyroid hormone and glucagon which produced maximal activation of PK were much lower than those required for maximal cAMP responses. Studies with charcoal indicated that these hormonal actions on PK reflected intracellular events rather than representing activation of the enzyme during tissue homogenization, due to release of sequestered cAMP. Thus, homogenization of tissue in charcoal prevented activation of PK by subsequent addition of exogenous cAMP, but did not lower enzyme activity ratios in homogenates of hormone-stimulated cortical slices. When PK was determined in the 20,000 g supernatant fraction of renal cortical slices incubated with the hormones, enzyme activity ratios also increased, but total enzyme activity declined. Lost activity was recovered by extraction of particulate fractions with 500 mM KCl or NaCl, results which implied particulate binding of activated PK. Activated soluble PK from renal cortex was bound equally well by intact, heat- and trypsin-treated renal cortical pellets and by intact and heated hepatic pellets. Accordingly, the apparent translocation of enzyme in hormone stimulated cortex does not necessarily represent binding of the activated PK to specific acceptor sites in the particulate cell fractions or constitute a physiologic hormonal action. Activation of renal cortical PK by increasing concentrations of salts suggests that the enzyme in this tissue resembles the predominant type found in heart.


Tissue T and B cell infiltration of primary and metastatic cancer. Immunofluorescent techniques were utilized to identify the types of infiltrating lymphocytes adjacent to human malignant tumors arising from a wide range of anatomic sites. 24 of 29 primary tumors and 5 of 8 metastatic lesions showed varying degrees of lymphocytic infiltration. T cells predominated in the infiltrates in primary tumors (mean 80%, range 50-100%) and this pattern was evident regardless of anatomic site or the presence or absence of metastatic spread. By contrast, B cells predominated at the margins of three of five tumor metastases. Mononuclear cells bearing the Fc receptor were not a prominent component of the infiltrates associated with either primary tumors or metastases, but tumor cell binding of fluoresceinated IgG aggregates was observed in 12 of 29 primary tumors. A significant reduction in peripheral blood T cell numbers occurred in a third of the patients studied. This decrease was not clearly related either to the extent of local tumor T cell infiltration or to the presence of disseminated disease. These preliminary findings provide a descriptive analysis of the local and systemic distributions of immunocompetent cells in cancer.


Pathophysiology of technetium-99m stannous pyrophosphate and thallium-201 scintigraphy of acute anterior myocardial infarcts in dogs. In 17 dogs with acute myocardial infarcts produced by ligation of the proximal left anterior descending coronary artery, a comparative study was made of myocardial scintigrams obtained with technetium-99m stannous pyrophosphate (99mTc-PYP) and thallium-201 (201T1), tissue levels of 99mTc-PYP and 201T1 uptake, histopathologic alterations, and regional myocardial perfusion measured with radioactive microspheres. 9 of the 10 hearts examined histologically had transmural infarcts with outer peripheral, inner peripheral, and central zones characterized by distinctive histopathologic features. A progressive reduction in myocardial blood flow was demonstrated between normal myocardium and the centers of the infarcts, and correlated well with progressive reduction in 201T1 upatke in the same regions. Marked 99mTc-PYP concentration occurred in areas with partial to homogeneous myocardial necrosis and residual perfusion located in the outer peripheral regions of the infarcts. The latter areas also were characterized by the presence of muscle cell calcification. The patterns of distribution of 99mTc-PYP and 201T1 explained the filling defects on 201T1 myocardial scintigrams and the doughnut patterns on 99mTc-PYP myocardial scintigrams in dogs with transmural infarcts. One dog with a subendocardial infarct had a small homogeneous area of activity on the 99mTc-PYP myocardial scintigram, and showed marked uptake of 99mTc-PYP in subendocardial areas of extensive necrosis and calcification still receiving some coronary perfusion. Thus, the data indicate that the status of regional myocardial perfusion is a key determinant for the occurrence of distinctive patterns of myocardial necrosis and for the scintigraphic detection of acute myocardial infarcts with 99mTc-PYP and 201T1.


Defect in intracellular killing of Staphylococcus aureus within alveolar macrophages in Sendai virus-infected murine lungs. Bacterial multiplication associated with virus infections is related to defects in in situ bactericidal (phagocytic) mechanisms of the lung. To determine whether the phagocytic defect was in bacterial ingestion and/or intracellular digestion, mice were infected with a sublethal dose of aerosolized Sendai virus and challenged 7 days later with a finely dispersed aerosol of Staphylococcus aureus. Groups of uninfected and virus-infected mice were sacrificed at 0, 6, 12, and 24 h after challenge, the lungs were perfused with formalin in situ, and the intra- or extracellular location of the bacteria was determined histologically. At 0 h, 49% and 51% of the staphylococci had an intracellular location in virus and nonvirus-infected lungs, respectively. With time, decreasing numbers of staphylococci were observed within the phagocytic cells of nonvirus-infected lungs, mostly as single organisms or in small clusters of less than four. In contrast, in focal area of virus-infected lungs, increasing numbers of phagocytic cells showed clumps of more than 25 bacteria/cell. These data demonstrate that virus-infected suppression of pulmonary antibacterial activity against S. aureus is related primarily to defects in intracellular processing mechanisms.


Interaction of prostaglandins and histamine with enzymes of cyclic AMP metabolism from guinea pig gastric mucosa. Prostaglandins (PGE1, PGE2, PGA1) and histamine have opposing effects on gastric HCl secretion, but we found that both stimulate adenylate cyclase activity in cell-free membrane preparations of guinea pig gastric fundic mucosa. The stimulatory effect of prostaglandins was found in this study to be specific and dose-dependent over a concentration range from 10(-7) to 10(-4) M. In similar preparations from antral regions of guinea pig gastric mucosa, the adenylate cyclase was stimulated only by PGE1, PGE2, and PGA1 and not by histamine. Maximum stimulating doses of PGE1, PGE2, or PGA1, and of histamine had an additive effect on the adenylate cyclase activity from fundic gastric mucosa. Metiamide, a histamine H2-receptor antagonist, inhibited the stimulation of fundic mucosa adenylate cyclase by histamine but did not interfere with the stimulation by prostaglandins. Cyclic AMP phosphodiesterase activity of guinea pig gastric mucosa was unaffected by PGE1 and PGE2 or by histamine, and was slightly depressed by PGA1. These results indicate that histamine and prostaglandins stimulate two different adenylate cyclase systems both present in guinea pig gastric mucosa tissue. Therefore, the known inhibitory effect of prostaglandins on gastric acid secretion is not related to the interference with the stimulation of the histamine H2-receptor-sensitive adenylate cyclase complex by histamine nor do prostaglandins accelerate cyclic AMP breakdown by cyclic AMP phosphodiesterase to reduce cyclic AMP levels.


The renin-angiotensin-aldosterone system in congestive failure in conscious dogs. The role of the renin-angiotensin-aldosterone system in the development of congestive failure has been assessed in the conscious dog by use of the nonapeptide converting enzyme inhibitor. Constriction of the pulmonary artery or thoracic inferior vena cava was maintained for 2 wk while daily measurements were made of plasma renin activity, plasma aldosterone, plasma volume, hematocrit, serum sodium and potassium concentrations, sodium and water balance, body weight, and arterial, caval, and atrial pressures. The initial response to constriction was a reduction in blood pressure, a rise in plasma renin activity, plasma aldosterone, and water intake, and nearly complete sodium retention. In the days after moderate constriction plasma volume and body weight increased (with development of ascites and edema); blood pressure, sodium excretion, plasma renin acvitity, and plasma aldosterone returned to normal. In animals in which blood pressure was not restored, plasma renin activity and plasma aldosterone remained elevated throughout the period of constriction. Single injections of converting enzyme inhibitor reduced blood pressure when plasma renin activity was elevated. Chronic infusion of the inhibitor in dogs with thoracic inferior vena caval constriction prevented the restoration of blood pressure and suppressed the rise in plasma aldosterone; sodium retention and volume expansion were less than in control experiments. Thus the renin-angiotensin-aldosterone system plays an essential role in the maintenance of blood pressure during the genesis of congestive failure. Initially, the restoration of blood pressure is dependent upon circulating angiotensin II; in the later stages, blood pressure is dependent upon the increase in plasma volume.


Effect of salicylates on histamine and L-histidine metabolism. Inhibition of imidazoleacetate phosphoribosyl transferase. In man and other animals, urinary excretion of the histidine and histamine metabolite, imidazoleacetate, is increased and that of its conjugated metabolite, ribosylimidazoleacetate, decreased by salicylates. Imidazoleacetate has been reported to produce analgesia and narcosis. Its accumulation as a result of transferase inhibition could play a part in the therapeutic effects of salicylates. To determine the locus of salicylate action, we have investigated the effect of anti-inflammatory drugs on imidazoleacetate phosphoribosyl transferase, the enzyme that catalyzes the ATP-dependent conjugation of imidazoleacetate with phosphoribosylpyrophosphate. As little as 0.2 mM aspirin produced 50% inhibition of the rat liver transferase. In vivo, a 30% decrease in the urinary excretion of ribosylimidazoleacetate has been observed with plasma salicylate concentrations of 0.4 mM. The enzyme was also inhibited by sodium salicylate but not by salicylamide, sodium gentisate, aminopyrine, phenacetin, phenylbutazone, or indomethacin. The last four drugs have been shown previously not to alter the excretion of ribosylimidazoleacetate when administered in vivo. Since both the drug specificity and inhibitory concentrations are similar in vivo and in vitro, it seems probable that the effect of salicylates on imidazoleacetate conjugation results from inhibition of imidazoleacetate phosphoribosyl transferase.


Exaggerated phosphaturic response to circulating parathyroid hormone in patients with familial X-linked hypophosphatemic rickets. To determine whether the phosphaturic response to circulating parathyroid hormone (PTH) is exaggerated in patients with familial x-linked hypophosphatemic vitamin D-resistant rickets (FHR), we examined the phosphaturic response to parathyroid extract (PTE) (administered intravenously in the posthypercalcemic state) in two unrelated adult hemizygotes with FHR. In these two patients whose plasma concentration of PTH was normal (determined by radioimmunoassay). neither vitamin D nor phosphate therapy had been given during the past 10 yr. Two normal men and a hypophosphatemic man with intestinal malabsorption, hypocalcemia, and osteomalacia served as control subjects. In all subjects, calcium gluconate was adminstered intravenously from 6 p.m. to 12 midnight at a rate that maintained the concentration of serum calcium at 13-15 mg/100 ml during the administration of calcium. When normocalcemia had recurred the next morning, and the plasma PTH concentration and urinary excretion of cyclic 3', 5'-AMP were reduced. PTE was administered intravenously at successively increasing rates of 0.1, 0.4, and 0.8 U/kg per h, each rate lasting 90 min. Minutes after the initiation of PTE in the affected hemizygotes, fractional excretion of filtered phosphate increased from negligible values to values strikingly greater than those of similarly studied control subjects and plateaued at strikingly greater values throughout further administration of PTE. This phenomenon of exaggerated phosphaturia could not be attributed to volume expansion, decreases in serum concentration of calcium during the study, differences in percent of administered calcium retained, or hemodynamic changes. Only the phosphaturic response to PTE appeared to be exaggerated. At any cumulative dose of PTE, urinary excretion of cyclic 3', 5'-AMP in the hemizogytes was indistinguishable from that of control subjects. The findings in this study suggest that in patients with FHR, circulating PTH is required for the genetically transmitted abnormality to be physiologically expressed as a reduction in net renal reabsorption of phosphate, and that this physiological expression of the genetic abnormality is expressed fully at normal or nearly normal circulating levels of PTH.


The regulation of plasma 18-hydroxy 11-deoxycorticosterone in man. 18-hydroxy 11-deoxycorticosterone (18-OH DOC), a weak mineralocorticoid, was estimated by a radioimmunoassay procedure after purification in 49 patients with hypertension and 38 normal control subjects. The sensitivity of the method was 2-4 pg; there was no detectable blank, and the precision was 9-10%. In normal subjects the absolute plasma levels were similar to those of aldosterone. ACTH administration produced a 23-fold increase, and sodium restriction resulted in a 4-fold increase (5.4+/-0.7-20.5+/-3.0 ng/dl). On the other hand, the plasma levels of 18-OH DOC declined by nearly 50% with upright posture or angiotensin II infusion. During both of these procedures, plasma aldosterone levels significantly increased. Patients with normal and low renin hypertension had similar changes in plasma 18-OH DOC levels with sodium restriction. However, the mean high sodium level in the normal renin essential hypertension group (11.6+/-1.6 ng/dl) was significantly greater (P is less than 0.001) than in the control group (5.4+/-0.7 ng/dl). In addition, at least 22% and perhaps as high as 37% of the hypertensive subjects had levels greater than the upper limits of normal on a high sodium intake. Differences between the groups were less impressive in the sodium-restricted studies. There were no significant differences in age, duration of hypertension, sodium balance, serum sodium, potassium, or blood urea nitrogen in those patients who had elevated levels of plasma 18-OH DOC. Patients with primary aldosteronism had levels within the normal range on both dietary intake. However, in contrast to the other groups there were no significant changes in the plasma levels with sodium restriction. Thus, a significant number of patients with essential hypertension presumably have an alteration in 18-OH DOC secretion.


Chemiluminescence and superoxide production by myeloperoxidase-deficient leukocytes. The role of superoxide anion- and myeloperoxidase-dependent reactions in the light emission by phagocytosing polymorphonuclear leukocytes has been investigated using leukocytes that lack myeloperoxidase, inhibitors (azide, superoxide dismutase), and model systems. Our earlier finding that oxygen consumption, glucose C-1 oxidation, and formate oxidation are greater in polymorphonuclear leukocytes that lack myeloperoxidase than in normal cells during phagocytosis has been confirmed with leukocytes from two newly described myeloperoxidase-deficient siblings. Although the maximal rate of superoxide anion production by myeloperoxidase-deficient leukocytes is not significantly different from that of normal cells, superoxide production falls off less rapidly with time so that with prolonged incubation, it is greater in myeloperoxidase-deficient than in normal cells. Chemiluminescence by myeloperoxidase-deficient leukocytes during the early postphagocytic period however is decreased. Light emission by normal leukocytes is strongly inhibited by both superoxide dismutase and azide, whereas that of myeloperoxidase-deficient  leukocytes, while still strongly inhibited by superoxide dismutase is considerably less sensitive to azide. Zymosan, the phagocytic particle employed in the intact cell system, considerably increased the chemiluminescence of a cell-free superoxide-H2O2 generating system (xanthine-xanthine oxidase) and a system containing myeloperoxidase, H2O2, and chloride. Light emission by the xanthine oxidase model system is strongly inhibited by superoxide dismutase and is not inhibited by azide, whereas the myeloperoxidase-dependent model system is strongly inhibited by azide but only slightly inhibited by superoxide dismutase. These findings suggest that light emission by phagocytosing polymorphonuclear leukocytes is dependent on both myeloperoxidase-catalyzed reactions and the superoxide anion, and involves in part the excitation of the ingested particle. These studies are discussed in relation to the role of the superoxide anion and chemiluminescence in the microbicidal activity of the polymorphonuclear leukocyte.


Fluorescent antibody responses to adenoviruses in humans. Specific IgG, IgA, and IgM immunoglobulin antibody responses to adenovirus infections were studied by the indirect immunofluorescent technique in six pairs of human sera obtained during acute and convalescent phases of the illness. In addition, 70 single specimens of sera showing adenovirus IgG antibody from different age groups from birth to the 60th year of life were titrated for the same antibody to adenovirus types 1, 2, 3, 5, and 7, and 170 serum specimens from the same age groups were screened for specific immunoglobulin antibodies against types 1 and 5. Specific immunoglobulin antibodies lacked type specificity and in acute infections measured heterologous antibody response as well. On the other hand, IgG antibodies detected in single specimens of sera by immunofluorescence correlate with surveys of the isolation of virus from patients and neutralizing antibody studies by other workers. Fluorescent antibodies appeared in all three fractions of the immunoglobulins in acute adenovirus infections. Although this technique may be used in the diagnosis of adenovirus infections there is no advantage compared to complement-fixation testing. However, the use of sera absorbed with group antigen may have a more useful place in serological epidemiology than in diagnostic work. In five pairs of sera obtained during acute and convalescent phases of adenoviral illness and in 70 random single specimens from different age groups, "T" antibodies were detected only in the IgG fraction. The paired sera did not show a significant rise to indicate the usefulness of "T" antibody study in diagnosis.


Serum beta-lipoprotein and other specific protein concentrations in patients with immunocytoma. Serum beta-lipoprotein and other specific protein concentrations were measured in 56 patients suffering from multiple myelomatosis, "benign" paraproteinaemia or Waldenström's macroglobulinaemia and in 56 control subjects. The mean level of B-lipoprotein in untreated patients with multiple myelomatosis and macroglobulinaemia was significantly lower than that of the controls. Patients who responded to chemotherapy showed a rapid return to normal of the beta-lipoprotein concentration, while the level remained unchanged in most of those who did not.


Membrane activity in histiocytic diseases of the skin. Study of the histiocytes from a large variety of skin diseases demonstrates a close relationship between the cell processes peculiar to those cells and their degree of phagocytic activity. The membrane structures, as well as the dense bodies localized in the peripheral cytoplasm, suggest a type of endocytosis distinct from pinocytosis and phagocytosis.


Regulation of bacterial motility by cyclic nucleotides and effect of biogenic amines. When assayed by a newly devised, simple and quantitative method, "motilometry", the motility of E. coli S-26 was found stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) and 8-hydroxy cyclic AMP as well as histamine, catecholamines and inhibited by cyclic guanosine 3':5'-monophosphate (cyclic GMP). The stimulation elicited by cyclic AMP or other biogenic amines was reversed by cyclic GMP. The experimental significance and implicaton of these findings are discussed.


Activation of cyclic AMP phosphodiesterase by a new vitamin E derivative. The effects of sodium alpha-tocopherol phosphate (TPNa), a new vitamin E derivative, on cyclic nucleotide phosphodiesterases from a soluble supernatant fraction of rat liver were investigated. TPNa produced a dose-dependent increase in cyclic AMP hydrolysis at a low substrate concentration (1 muM cyclic AMP), whereas the compound inhibited the hydrolytic activity at a high substrate level (100 muM cyclic AMP). Cyclic GMP phosphodiesterase activity was suppressed by TPNa regardless of the substrate concentration. The addition of TPNa did not change the apparent Km value (50 muM) of cyclic AMP phosphodiesterase at low substrate level (less than 5 muM). In contrast, at higher substrate concentration, the concave downward curve observed in a Lineweaver-Burk plot became straight in the presence of TPNa. Low concentrations of cyclic GMP, which are known to activate cyclic AMP hydrolysis, showed an additive effect on cyclic AMP phosphodiesterase only when a submaximal concentration of cyclic GMP was present in addition to TPNa. These and other data suggest that TPNa modifies cyclic AMP phosphodiesterase in all allosteric fashion.


Effect of acetylcholine on glycogen phosphorylase activity and cyclic nucleotide content in isolated perfused rat hearts. Acetylcholine (1muM) increased cyclid GMP content in paced perfused rat hearts within 15 sec., with peak content occurring at 1 min. No effect of acetylcholine on cyclic AMP content, phosphorylase activity or glycogen synthase was observed. Epinephrine (1muM) infusion increased both cyclic AMP content and phosphorylase, but did not alter cyclic GMP content or glycogen synthase activity. When acetylcholine was infused during the second min. of a 2 min. infusion of epinephrine, the cholinergic agent increased cyclic GMP and reduced the stimulated phosphorylase activity and elevated cyclic AMP.


Cyclic nucleotide levels during carbachol-induced smooth muscle contractions. Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cyclic GMP levels could be detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cyclic GMP levels could be detected during carbachol-induced contractions (i.e. guinea pig myometrium and taenia coli) the contractions appeared to precede the cyclic GMP increases by several seconds. No significant changes in cyclic AMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Thus, changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.


Bronchiolitis and asthma: possible common pathogenetic pathways. A discussion of possible mechanism for virus-induced wheezing in normal and asthmatic children is presented. Presently attractive theories for immune injury in viral bronchiolitis include those which depend on pathology induced by types 1, 3, and 4 of Gell and Coombs. The continuum of viral bronchiolitis with virus-induced wheezing in young children who are labeled "asthmatic" argues for some common mechanism of bronchiolar obstruction.


Study of the arrangement of crystallites in gamma-irradiated human enamel by electron paramagnetic resonance. The arrangement of tooth enamel microcrystals has been studied on CO3-3 bound electrons by electron paramagnetic resonance. It was found that noncarious human maxillary central incisors have a greater degree of alignment of tooth enamel microcrystals than the carious ones. The outermost surface layer of enamel showed a greater crystallite degree of alignment than other parts.


[Effects of methotrexate on cervico-vaginal cytology]. Disturbances of folic acid metabolism can bring about the appearance of characteristic displastic cells, FD cells, in the cervico-vaginal epithelium. A study of the colpocytogram in a series of patients who had trophoblastic tumours treated with methotrexate shows that the administration of an antifolic agent can provoke under certain conditions the appearance of FD cells or an increase in their number when they are present before chemotherapy has been started. These cells are in a variable percentage of cases, transitory in nature but when methotrexate treatments are repeated they often return as a regular feature. The cytomorphologic aspect of these FD cells when they appear after chemotherapy is identical to those that exist spontaneously in certain deficiency conditions occuring in pregnancy and the puerperium.


Cytochemical model system for microsomal rat liver glucose-6-phosphate. A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.


Studies of experimental rhinovirus type 2 infections in polar isolation and in England. After five months of total isolation a wintering party of seventeen British Antarctic Survey (BAS) personnel was inoculated under double blind concitions with placebo, or rhinovirus type 2 which had been propagated in tissue culture. The clinical and virological responses of these subjects were compared with those of volunteers in England who received a similar dose of the same strain. The virus used was apparently partly attenuated for man; at the dosage used its effects in England were similar to a smaller dose of an unattenuated strain, but in the Antarctic it caused relatively severe infections. Both the symptoms and the laboratory evidence of virus infection appeared to be more pronounced in the BAS subjects than in the volunteers in England who received the same challenge. In the former group the infection readily spread to those who were originally given placebo. In the BAS subjects serum antibody titres were well maintained during the isolation period but a significant fall in nasal immunoglobulin concentration was recorded during the 5 months of isolation after the virus challenge. Possible mechanisms for the increased sensitivity to rhinovirus of subjects who have been totally isolated in a small closed community are discussed.


The role of sheep and goats in the epizootiology of foot-and-mouth disease in Kenya. The pathogenicity of two bovine field strains of virus for indigenous goats was examined in the laboratory. The goats failed to develop clinical disease or become virus carriers although the majority showed a definite immune response. A field survey in a foot-and-mouth disease enzootic area showed that the indigenous sheep and goat populations were frequently exposed to infection as evidenced by a high proportion of sero-positive animals but the incidence of virus carriers was very low in goats and no carriers were detected in sheep.


Growth of foot-and-mouth disease virus in the upper respiratory tract of non-immunized, vaccinated, and recovered cattle after intranasal inoculation. Non-immunized, vaccinated, and recovered cattle were inoculated intranasally with various doses of foot-and-mouth disease virus. Samples of oesophageal-pharyngeal (OP) fluid were taken periodically for up to 7 days after inoculation and virus titres of these samples were plotted as pharyngeal virus growth curves. In non-immunized cattle, the length of the lag period and of the growth period were inversely proportional to the dose of virus given. Maximum titres were observed when clinical signs were first detected. Three of the 10 cattle studied had virus growth rates that were lower than rates of others given the same dose of virus, and clinical signs appeared later than expected in these three cattle. Cattle vaccinated with an inactivated virus oil-adjuvant vaccine had pharyngeal virus growth curves that were similar to those obtained from non-immunized cattle for 30 h. after inoculation. Titres of virus in OP fluid samples taken 2-7 days after inoculation were substantially lower in cattle with a high pre-exposure serum mouse protection index than titres from partly-immunized or non-immunized cattle. Nine of 14 cattle had detectable but reduced virus growth after intranasal inoculation with homologous virus. Five recovered cattle inoculated with heterologous virus reacted similarly to non-immunized animals.


Immunologic mechanisms in the pathogenesis of virus-induced murine leukemia. III. Target cell specificity of autoreactive thymocytes. Thymocytes from preleukemic mice persistently infected with Moloney murine leukemia virus (MuLV-M-carriers) were vigorously autoaggressive toward normal syngeneic target cells; they exhibited a graded response to allogeneic cells, but they spared xenogeneic cells or syngeneic cells infected with MuLV-M or MuLV-G (Gross). Syngeneic target cells infected with nononcogenic lymphocytic choriomeningitis virus (LCMV), or transformed by the chemical carcinogen 3-methylcholanthrene were not similarly spared. This phenomenon, apparently induced by MuLV-M, is not associated with all persistent virus carrier states. Thymocytes from mice persistently infected with LCMV or with the lactic dehydrogenase virus (LDHV) failed to demonstrate an autoaggressive behavior. That transplantable lymphoma cells (derived from MuLV-M-carriers) were autoreactive in a pattern similar to thymocytes from preleukemic mice suggests a unique role for MuLV in the events leading from altered recognition of "self" to lymphoma.


Appearance of IgG (Fc) receptor(s) on cultured human fibroblasts infected with human cytomegalovirus. Cultured human diploid fibroblasts (WI-38) after infection with human cytomegalovirus (CMV) but not when uninfected, could hemadsorb sheep red blood cells (SRBC) coated with rabbit anti-SRBC IgG. The adsorption of IgG-coated SRBC to virus-infected cells was completely abolished if the tests were carried out in the simultaneous presence of rabbit antiserum elicited against CMV. Normal sera of rabbit or human origin as well as purified human IgG but not Fab fragment of human IgG could also abolish the binding of sensitized SRBC to CMV-infected fibroblasts. Active metabolism on the part of CMV-infected fibroblasts proved to be an important requisite for demonstrating binding of sensitized SRBC to their surfaces. By using an indicator Staphylococcus aureus to which rabbit antiserum against normal human IgG, IgM, or IgA was bound via Fc fragments, evidence has been obtained which suggests the existence of receptor(s) on CMV-infected WI-38 cells that react specifically with Fc region of human IgG.


Enhancement of early human E rosette formation by cholinergic stimuli. The effects of the cholinergic stimuli carbamylcholine (carbachol) and dibutyrl cyclic guanosine monophosphate (DBCGMP) were determined on both 'early' and 'total' E rosette formation. Ficoll-Hypaque-separated lymphocytes were preincubated with either carbachol or DBCGMP over a 10(-3) M to 10(-13) M dose range. Both agents significantly enhanced 'early', but not 'total' E rosette formation. Peak enhancement above control values occurred at 10(-7) M (72%) and 10(-9) M (69%) for carbachol and 10(-5) M (70%) and 10(-7) M (70%) for DBCGMP. Kinetic studies showed a rapid onset of enhancement (2.5 min) for carbachol, whereas DBCGMP required 15 min for significant enhancement to occur. The muscurinic nature of carbachol enhancement of E rosettes was demonstrated. Atropine at 10(-7) M completely abolished the carbachol effect while showing little inhibition of the DBCGMP effect on rosette formation. These studies indicate that the cholinergic stimuli carbachl and DBCGMP significantly enhance the 'early' E rosette former in man. Human T lymphocytes appear to have functional cholinergic receptors that can be blocked by the muscurinic antagonist atropine. The role of the cyclic nucleotides and their stimulants on the immune system is incompletely understood, but it would appear that they are extremely important in the differentiation and function of the T lymphocyte. E rosette formation may be a useful model in man for studying the effects of the cyclic nucleotides on the human T lymphocyte.


Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors. Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.


Chromatographic separation of gram quantities of immunoglobulins from porcine colostrium against transmissible gastroenteritis virus. Similar immunoglobulin (Ig) classes were obtained from porcine colodtral whey by either column or batch chromatographic procedures; a stepwise buffer elution technique was used. Specific transmissible gastroenteritis virus neutralizing antibody was found in the 4 major fractions eluted comprising of IgG1, IgG2, IgA, and IgM. The IgG1, and IgG2 were essentially homogeneous, and the IgA- AND IgM-rich fractions had to be recycled several times through Sephadex G-200 to obtain pure IgA and IgM that had specific virus neutralizing activities per mg of protein of 342.1 and 302. 4, compared with 7.6 for IgG. By a combination of the batch chromatographic procedures and gel filtration, gram amounts of specific Ig could be fractionated from the same colostrum.


High density lipoprotein (HDL) polymorphisms in rabbit. I. A comparative study of rabbit and human serum high density lipoprotein. Different classes of rabbit serum lipoprotein were prepared by ultracentrifugal flotation at densities 1-006, 1-063 and 1-21 g/ml. Agarose gel electrophoresis on rabbit whole serum and the serum fractions with different densities showed that this technique separates the different lipoprotein classes reasonably well. The electrophoretic mobility of the different lipoprotein classes of rabbit serum seems to be similar to that of the human lipoproteins, with the exception of alpha1-lipoprotein which had a greater mobility than human alpha1-lipoprotein. The chemical composition of rabbit high density lipoprotein (HDL)p was fairly similar to that of human HDL although the former seems to be richer in triglycerides. HDL was, after isolation by ultracentrifugal flotation at density 1-21, delipidated and submitted to gel filtration on Sephadex G-200 in 8 M urea. The major protein fraction of rabbit apo HDL corresponds in elution volume to that of the major fraction of human apo HDL, apoA-I. A protein fraction corresponding to human apoA-II does not seem to be present in rabbit HDL in demonstrable amounts. The rabbit protein fraction sometimes appearing in the area corresponding  to human apoA-II could not be found to be affected by the reduction and alkylation method after which human poA-II splits into two identical chains.


Regulation of cell cycles.

Regulation of cell cycles. Of the major achievements in cell biology during the last 25 years, none is more important than the understanding of regulation of cell cycles. In 1953 two fundamental observations concerning DNA were made. Watson and Crick suggested that the three-dimensional structure of DNA exists as a double helix with specific base pairings, and Howard and Pelc observed that DNA is replicated during a specific phase in the mitotic cycle. Thus developed the theory of cell cycles. Next, investigators explored which events occur during each phase of the cycle and what controls the readout of the genes of proliferation or differentiation. In 1961, Jacob and Monod proposed that for prokaryotic cells the operon is the mechanism which controls the readout of the genes; and by the end of the 1960s, several investigators had defined the role of cyclic AMP and its mechanism of action at the gene level. The control mechanisms of eukaryotic cells are less well defined. Basically there are two types of regulatory molecules: those that arrive at the cell surface and send messages inside the cell; and those that enter the cell, bind to receptors, and then enter the nucleus to interact with the genes. During the past five to ten years, the cell surface and its receptors have received considerable attention as the recognition and control areas for cell proliferation and differentiation, and currently the role of the cyclic nucleotides and prostaglandins is being investigated. Various model systems are now available for detailed studies of these control mechanisms.


Viral diseases of the skin, 1975: a 25-year perspective. Most of the major advances in modern virology during the past 25 years have been due principally to the development of refined laboratory techniques and tools and have provided a fund of new knowledge and information about the nature of viral infection and pathogenesis. One group of viruses of interest to dermatologists, the herpesviruses, is undergoing intensive biochemical investigation to determine whether it is carcinogenic. As a result of the success of the World Health Organization's campaign to eradicate smallpox, it is predicted that by the end of 1976, smallpox will have been eradicated. Other viruses of dermatologic interest which are now being studied include the agents of warts, molluscum contagiosum, cat-scratch disease, and enteroviruses. Current research in the field of viral chemotherapy may provide the basis for successfully treating these diseases in the future.


The culture of skin. A review of theories and experimental methods. Two main criticisms can be leveled against the standard methods of skin culture: they are poorly quantifiable and the cultured cell populations are heterogeneous. A new technique based mainly on enzymatic dissociation allows specific cell types to be extracted from the skin before cultivation. In this way, separate cultures of epidermal keratinocytes and dermal fibroblasts can be obtained from the same piece of skin. These purified systems have been used to study the kinetics of epidermal cell growth and to quantify the effect of various chemically defined substances on the growth and differentiation of keratinocytes. With further refinements in technique, purified populations of melanocytes can be extracted. The co-culture of pigmented melanocytes with albino keratinocytes has been proposed as a model to study pigment donation in vitro. The usual organ culture technique, including the use of large explants of skin immersed in the culture fluid, has been modified to show that adult human skin partially regenerates in vitro and that mitotic activity goes on for months in the regenerated epidermis. The use of nucleic acid hybridization techniques, combined with skin cell cultures from human tumors, opens new avenues of research on human cancer.


Evaluation of 6-azauridine and 5-iododeoxyuridine in the treatment of experimental viral infections. The potential antiviral activity of 6-azauridine and 5-iododeoxyuridine was evaluated in a coordinated study at five institutions. Experimental models in five species, the mouse, rabbit, swine, cat, and ferret, were established with use of 10 viruses: Herpesvirus hominis types 1 and 2, murine cytomegalovirus, vaccinia virus, Shope fibroma virus, transmissible gastroenteritis virus, swine influenza virus, feline viral rhinotracheitis virus, feline panleukopenia virus, and ferret distemper virus. Criteria for selection were: (1) representation from a number of major groups of viruses, (2) reproduction of natural routes of infection, and (3) simulation of potentially treatable viral infections of man. Antiviral activity was observed for 5-iododeoxyuridine in H. hominis infections in hairless mice and influenza in swine, and a slight degree of efficacy was noted in rabbits infected with Shope fibroma virus. Toxicity was also observed in most of the experimental models. There was a suggestion of antiviral activity with 6-azauridine in swine infected with transmissible gastroenteritis virus; however, enhancement of disease and some toxicity were seen in most of the other models. Efficacy of these two compounds was not well substantiated by these studies.


In vitro stimulation of lymphocytes of donors seronegative for Epstein-Barr virus by preparations of inactivated Epstein-Barr virus. Heat-inactivated preparations of Epstein-Barr virus stimulated human lymphocytes as assayed by incorporation of [(3)H]thymidine. The inactivated Epstein-Barr virus stimulated the lymphocytes of all five seropositive donors, 11 of 14 seronegative donors (aged eight to 26 years), and none of 15 neonates. Control antigens prepared from a human lymphoid cell line devoid of the Epstein-Barr virus genome did not stimulate the lymphocytes of seronegative donors. Fetal calf serum at the concentration used for suspension of Epstein-Barr virus did not stimulate or only minimally stimulated the lymphocytes of seronegative donors. The reactivity of the histocompatibility antigens found on human lymphocytes was abolished by procedures used for inactivation of the virus.


Treatment of infections due to Herpesvirus in humans: a critical review of the state of the art. Results of experimental trials with antiviral agents in humans have varied from encouraging to controversial to negative, usually as a result of the difficulty in defining the true therapeutic index (ratio of efficacy to toxicity) of toxic drugs for the treatment of diseases that are potentially severely debilitating or lethal. Reasons for current difficulties relate mainly to inadequacies of preclinical studies and the lack of appropriate controls. The inadequacies include poor definition of the effect of drugs or viruses on cellular metabolism, incomplete pharmacologic studies in animals or humans, and, because of the latter, inappropriate animal models. In human trials, historical data have often been used instead of true controls because of the presumed severity of candidate diseases. Use of such data led to a false impression of drug efficacy, an impression later refuted when proper control studies demonstrated that the range of disease was much greater than had been previously supposed. Data bearing on these points for the most commonly employed experimental compounds (cytosine arabinoside, adenine arabinoside, and 5-iodo-2'-deoxyuridine) are contrasted to highlight difficulties as well as to provide perspectives for antiviral chemotherapy of herpesvirus infections.


Effect of a topical interferon inducer on rhinovirus infections in volunteers. Six controlled trials of a topical interferon inducer, N',N'-diotadecyl-N',N'-bis-(2-hydroxyethyl) propanediamine (CP 20961), against rhinovirus infection in volunteers were reviewed. Controls were the drug vehicle (which consisted of a mixture of polysorbate-80 in gylcerol and saline), saline only, and oral lactose. Multiple doses of the inducer given before challenge in any of three regimens reduced the symptomatic response to viral infection. Treatment with three doses of drug the day before challenge enhanced the interferon response to virus; four days of treatment with either the drug or its vehicle before challenge produced a refractoriness to further interferon induction. Both the drug and its vehicle were capable of inducing nasal interferon. When interferon was present at the time of viral inoculation, symptoms were reduced with or without an increase in the interferon response to infection. The rates of infection and illness were increased after a single dose of drug and decreased after a single dose of the vehicle alone.


Four compounds active against rhinovirus: comparison in vitro and in volunteers. Four unrelated compounds active against rhinovirus were compared in tissue culture, and three of them were used in volunteers challenged with rhinovirus. The compounds were the triazino-indole SKF 40491, the substituted oxadiazole GLR9-338, the imidazo-thiazole RP L9326, and the guanidine derivative ICI 73,602. The abilities of these compounds to reduce the yield of rhinovirus types 3, 4, 9, and 31 from HeLa cells or fibroblasts were compared, and a sensitive serotype was chosen for each challenge experiment in humans. In doubleblind studies volunteers received intranasal medication before and after the challenge. Daily scoring of symptoms and titration of virus in nasal washings showed that subjects treated with SKF, GL, and RP all shed less virus than their corresponding placebo groups, significantly so in the cases of GL and RP. Clinical reactions were also less severe in volunteers treated with RP.


Sensitivity of rhinoviruses to human leukocyte and fibroblast interferons. Rhinoviruses were tested for sensitivity to human interferon from two sources: human leukosytes induced with Sendai virus and human diploid fibroblasts induced with polyriboinosinic-polyribocytidylic acid. Twenty-five serotypes of rhinovirus were tested against fibroblast interferon in HeLa cells, and five serotypes were evaluated in fibroblast cultures against fibroblast and leukocyte interferons. Sensitivity varied widely in the HeLa cell assay; rhinovirus type 10 was inhibited by approximately 20-40 units of interferon, whereas rhinovirus type 15 was not inhibited by 5,120 units; the significance of this observation is unclear. In contrast, when fibroblast cultures were used in the assay system, five selected serotypes, including rhinovirus types 10 and 15, had similar levels of sensitivity (0.5-5.0 units) to fibroblast or leukocyte interferon.


Photodynamic inactivation in recurrent infections with herpes simplex virus. Photodynamic inactivation with neutral red (a heterotricyclic dye) and light was evaluated in a placebo-controlled study of 170 episodes of recurrent infection with herpes simplex virus in 96 patients. The technique inactivated herpes simplex virus in vitro. However, no beneficial effect, either on the rate of resolution of herpetic lesions or on the interval to subsequent recurrences, was observed in treated patients. These negative results should not deter further investigation of photodynamic inactivation as a potential treatment for herpes simplex virus infections, but the clinical application of this form of therapy should be limited to properly controlled studies until its efficacy is proven.


Genital herpetic infection in men and women: clinical course and effect of topical application of adenine arabinoside. Sixty-three episodes of genital herpetic infection in 55 men and 45 episodes in 42 women were randomly assigned to topical treatment with 3% adenine arabinoside, topical treatment with placebo ointment, or no therapy. In addition, 10 episodes in women who were not randomly assigned to therapy were evaluated and followed. Clinical evaluation and viral cultures were done on day 3, day 8, and weekly thereafter until the lesions had healed. The natural clinical course of genital herpetic infection was defined in patients given placebo or no therapy. Patients with a history of previous genital herpetic infection had significantly less pain, few lesions, and shorter duration of illness and viral shedding than patients who had no history of previous genital infection with Herpesvirus. An unexpected finding was that 87% of women experiencing their first episode of genital herpetic infection had cervical cultures positive for Herpesvirus hominis, whereas only 4% of women with recurrent herpetic infection had a positive cervical culture. Treatment with 3% adenine arabinoside did not influence the course of either primary or recurrent genital infection with Herpesvirus.


Interferon in treatment of dendritic keratitis in humans: a preliminary report. Seventy-three patients with herpetic epithelial keratitis were randomly divided into three groups and were treated by local applications of human leukocyte interferon, thermocautery plus human leukocyte interferon, or thermocautery plus mock human leukocyte interferon to obtain information on whether therapy with human leukocyte interferon can serve as a substitute for mechanical debridement of the involved epithelium or can improve the results when given as additional therapy. Fifty-five patients (75%) yielded herpes simplex virus before treatment, and only the results in these patients with keratitis of proved viral etiology were included in the analysis. These results indicated that mechanical debridement cannot be replaced with local application of human leukocyte interferon (3-4 X 10(4) units per day). When given in addition to thermocautery, human leukocyte interferon may have been of some value in a number of patients. However, the results were not statistically significant. A more potent dosage of human leukocyte interferon might provide better results.


Adenine arabinoside for therapy of herpes zoster in immunosuppressed patients: preliminary results of a collaborative study. Eighty-seven immunosuppressed patients (53 with localized and 34 with disseminated herpes zoster) from 10 institutions were enrolled in a controlled therapeutic trial of adenine arabinoside. A crossover design was employed; thus, 47 patients received drug for five days and then received placebo, and 40 were given the two substances in the opposite order. Resolution of acute pain and the cutaneous lesions were graded during a 10-day observation period. During the initial five-day period, treated patients showed a statistically significant resolution of pain and cutaneous lesions. Surprisingly, in many untreated patients, natural resolution occurred during this period so that crossover data could not be adequately assessed. Effects on visceral disease also could not be judged, because such disease was uncommon. Ratings of late complications such as postherpetic neuralgia were confused by the crossover design. Toxicity posed no problem. The data further emphasized the potential usefulness of adenine arabinoside as an antiviral chemotherapeutic agent, but also clearly indicated the need for a double-blind study to define this usefulness and to determine how it can most practically be used, if the risk-benefit factor remains favorable.


Toxicity of adenine arabinoside in humans. Six forms of reversible adverse reactions to adenine arabinoside (vidarabine) were observed in a two-year period among 42 patients (19 of whom had lymphomas, leukemias, or other malignancies) who were treated for complicated infections with varicella-zoster or herpes simplex virus. Six patients received placebo. Ten patients received 10 mg of adenine arabinoside/kg per day; three received 15 mg/kg; 22 received 20 mg/kg; and one received 30 mg/kg. Patients were treated (by continuous intravenous injection) for an average of seven days. Toxic effects were nausea and vomiting, weight loss, weakness (often with impaired ambulation), megaloblastosis in erythroid series in bone marrow, tremors five to seven days after the start of therapy (including tremors in one patient with abnormal electroencephalograms that were consistent with toxic-metabolic encephalopathy), and thrombophlebitis at the intravenous site. Side effects clearly predominated in patients who received 20 mg/kg per day. Therefore, treatment with 10 mg/kg per day appears preferable until the relation of toxicity to dosage level can be clarified.


Effect of human exogenous leukocyte interferon in cytomegalovirus infections. Human leukocyte interferon was injected into nine patients with cytomegalovirus infections; four of these patients were congenitally infected, and five had acquired infections. In three patients viruria was completely inhibited. In five patients viral excretion in the urine was only transiently inhibited. Viremia was not significantly suppressed. The lymphocyte response to phytohemagglutinin was suppressed in two patients.


Effect of leukocyte interferon on urinary excretion of cytomegalovirus by infants. Five infants with symptomatic cytomegalovirus infections and high urinary titers of cytomegalovirus were treated with interferon in doses of 1.7-3.5 X 10(5) reference units/kg per day for seven to 14 days. Six courses of treatment were given. One of two infants treated with the largest dose of interferon had transient suppression of viruria; no suppression was noted during the other five courses of treatment. Adverse effects noted included a low rate of weight gain, a transient elevation of serum aspartate aminotransferase and fever. Further trials are needed to determine whether larger doses of a more purified preparation of interferon will eliminate viruria in infants with sytomegalovirus infection, but careful assessment of toxicity will be necessary in this population of patients.


Effective antiviral prophylaxis and therapy by systemic application of human interferon in immunosuppressed monkeys. Rhesus monkeys treated with antilymphocytic globulin were infected with vaccinia virus or with Medical Lake macaque Herpesvirus. At the sites of inoculation of vaccinia virus, animals not treated with interferon developed typical pustulous reactions and then smaller secondary lesions. Human leukocyte interferon (5 X 10(5) international units per day) injected intramuscularly, either from one day before to three days after infection or for seven days beginning on the first day of primary reaction, suppressed the development of secondary lesions but did not influence the primary reaction. Infection with Herpesvirus caused a vesicular rash similar to that of human varicella. Through prophylactic treatment with human leukocyte interferon, the disease was completely averted in two of four monkeys, and in the other two the rash was delayed. Therapy initiated when the first lesions appeared prevented the enlargement and generalization of lesions in all of four monkeys. Formation of serum antibodies was only slightly influenced by human leukocyte interferon. The results suggest that systemic treatment with interferon, begun either prophylactically or therapeutically with the first symptoms, can be effective in immunosuppressed patients.


Immunopotentiation and antiviral chemotherapy in a suckling rat model of herpesvirus encephalitis. Encephalitis was induced in 10-day-old Wistar rats by intraperitoneal injection of approximately 100 50% tissue culture infective doses of herpes simplex virus type 2. Treatment regimens included immunopotentiation with levamisole and combined therapy with levamisole and an antiviral agent, adenine arabinoside. Rats treated with levamisole alone had significantly higher rates of survival than placebo-treated controls 14 days after injection of virus. Combination therapy with levamisole and adenine arabinoside prolonged survival, but there was no significant difference between treated animals and controls given placebo. Because adenine arabinoside inhibits the beneficial effect of levamisole in this model, antiviral chemotherapy in conjunction with immunopotentiation should be used with caution in humans. Further studies will be necessary to determine the value of immunopotentiation therapy in the treatment of life-threatening viral infections.


Experimental encephalitis caused by herpes simplex virus: comparison of treatment with tilorone hydrochloride and phosphonoacetic acid. A mouse model of encephalitis caused by herpes simplex virus was used to compare the antiviral activity of tilorone hydrochloride with that of phosphonoacetic acid. These compounds were also administered simultaneously to determine whether the combination had a synergistic effect. Rates of survival and concentrations of virus in brain were used as criteria for judging the effectiveness of treatment. The fatal course of encephalitis was not altered by any treatment protocols in which tilorone hydrochloride was used alone. Four days of treatment with phosphonoacetic acid resulted in a long-term survival rate of about 15% of the infected, treated animals, and extension of this therapy for an additional three days resulted in an overall survival rate of about 35%. No increase in survival rate was obtained by use of phosphonoacetic acid and tilorone hydrochloride in combination. The concentration of virus in the brains of tilorone hydrochloride treated animals did not differ significantly from that in the untreated, infected control animals. Treatment with phosphonoacetic acid resulted in a reduction in titer of virus in brain and in an increased rate of survival.


Intranasal challenge of mice with herpes simplex virus: an experimental model for evaluation of the efficacy of antiviral drugs. An experimental model of herpetic infection based on intranasal challenge of 12-day-old mice with herpes simplex virus (type 1) has been developed for assessment of the efficacy of a variety of antiviral compounds with clinical potential: cytosine arabinoside, adenine arabinoside, iododeoxyuridine, ribavirin, chloriteoxidized oxyamylose, polyriboinosinic-polyribocytidylic acid, and interferon. The model employed is reminiscent of herpetic encephalitis in humans in both the portal of entry (nasopharyngeal cavity) and the mode of transmission (nerve route) of the virus to the target organ (brain). The mortality rate from viral infection was significantly reduced (greater than or equal to 30%) by the following treatment regimens: cytosine arabinoside, adenine arabinoside, iododeoxyuridine, and ribavirin, administered daily for seven consecutive days starting immediately after inoculation of virus, at dosage levels of 4-20 mg/kg, 20-100mg/kg, 100mg/kg, and 20-100 mg/kg, respectively; and chlorite-oxidized oxyamylose, polyriboinosinic-polyribocytidylic acid, and mouse interferon, administered 24 hr before viral challenge, as single doses of 100-500 mg/kg, 20mg/kg, and 10(7)-10(8) international reference units/kg respectively. Similar doses of polyriboinosinic-polyribocytidylic acid and mouse interferon administered after inoculation of virus did not alter the final mortality rate.


Effective antiviral chemotherapy in cytomegalovirus infection of mice. Both murine and human strains of cytomegalovirus were shown to be sensitive to the antiviral effects of adenine arabinoside and phosphonoacetic acid in tissue culture. In mice with lethal cytomegalovirus infections, treatment with adenine arabinoside (either 500 mg/kg intraperitoneally once daily or 250 mg/kg intraperitoneally twice daily for seven days) failed to reduce the mortality rate or to decrease the mean number of days until death. In contrast, treatment with the same dosage regimen of phosphonoacetic acid significantly reduced the mortality rate and decreased the mean number of days until death even when therapy was delayed for 24 hr. Although early treatment with phosphonoacetic acid resulted in a marked reduction in viral titers in the brains and lungs of infected mice, the major reason for efficacy appeared to be complete inhibition of viral replication in the liver. The observation that titers of complement-requiring neutralizing antibody were significantly lower in treated animals than in untreated controls is further evidence of successful therapy. Treatment of a nonlethal cytomegalovirus infection with phosphonoacetic acid beginning 48 hr after viral challenge resulted in elimination of clinical signs of illness and reduction in viral titers in tissues. These results indicate that phosphonoacetic acid is successful in the treatment of murine infections with cytomegalovirus; they suggest that this drug should be considered for potential use in serious cytomegalovirus infections in humans.


Prophylactic control of simian hemorrhagic fever in monkeys by an interferon inducer, polyriboinosinic-polyribocytidylic acid-poly-L-lysine. A nuclease-resistant complex of polyriboinosinic-polyribocytidylic acid containing poly-L-lysine and carboxymethylcellulose was used as prophylactic treatment of simian hemorrhagic fever in rhesus monkeys. This infection has proven uniformly fatal to more than 50 monkeys. Treatment 8 hr before infection and repeatedly thereafter completely prevented the development of viremia and death. Untreated animals died before development of antibodies to the virus. None of the treated animals developed antibody to the virus, a fact which suggested that viral growth was essentially completely blocked by the compound.


Measurements of the prevalence of viral infections. Viral diseases exert their major impact through morbidity, impairment of personal health, loss of time at work and school, and cost of medical care. Relatively few of the known viruses cause a significant number of deaths; influenza, childhood viral pneumonia, and hepatitis are the only viral diseases causing more than 1,000 deaths per year. Data based on the National Health Interview Survey of the National Center for Health Statistics show that the common cold annually causes 35.6 acute illnesses per 100 persons. The data reported by the National Therapeutic Disease Index (on the basis of visits by patients to a sample of 1,500 private physicians) show that influenza and other acute respiratory conditions account for about one-third of all visits to physicians. Nearly all viruses first infect humans in infancy and childhood, with a relatively low fatality rate but with frequent episodes of illness.


Persistence of the viral genome in interferon-treated cells infected with oncogneic or nononcogenic viruses. In AKR mouse cells chronically infected with a murine leukemia virus, treatment with interferon for nine days resulted in sustained inhibition of extracellular production of murine leukemia virus but no inhibition of viral intracellular p30 antigen or of reverse transcriptase. Removal of interferon resulted in rapid reversal of these effects. Interferon-treated mouse L-cells were infected with high multiplicities of vesicular stomatitis virus or herpes simplex virus type 1. Infectious virus and intracellular viral antigen were rapidly eliminated from the interferon-treated cultures infected with herpes simplex virus. In cultures infected with vesicular stomatitis virus, titers of virus remained low in interferon-treated cells, but after about two weeks they rose rapidly and the cultures were destroyed. If treatment with interferon was reinstituted as late as nine days after primary infection, infectious vesicular stomatitis virus was eliminated, and there was no evidence for survival of the viral genome in these cultures. In the cultures infected with murine leukemia virus, inhibition of production of virus by treatment with interferon was possible, but the viral genome was not eliminated. In cells acutely infected with vesicular stomatitis virus or herpes simplex virus, however, the viral genomes were apparently eliminated from cultures treated with interferon.


Influence of interferon on the synthesis of virus particles in oncornavirus carrier cell lines. IV. Relevance to the potential application of interferon in natural infectious diseases. That interferon reduced the release of C-type oncornavirus particles by chronically infected mouse cells was shown by radiolabeling of the particles with uridine or amino acids and by determination of particle-associated reverse transcriptase. The number of released particles, as determined by direct electron microscopic enumeration, was reduced to a lesser extent. In contrast, interferon failed to affect the number of budding particles and caused a slight increase in the number of completed particles present in the microspace contiguous to the cell membranes. A working hypothesis is that, in the presence of interferon, C-type particle assembly and release are slowed but not arrested; sizable numbers of particles continue to be assembled and released. Some of these particles may be defective in one or more proteins, such as reverse transcriptase or proteins necessary for final release. These in vitro data justify speculation that, in vivo, interferon may be expected to reduce tissue damage due to antigen-antibody complex formation, but not damage due to sytolytic immune attack on cells carrying the antigens.


Localization fo the gene AVG for the antiviral expression of immune and classical interferon to the distal portion of the long arm of chromosome 21. The responses of normal fibroblasts and of fibroblasts trisomic and monosomic for chromosome 21 to exogenously administered virus-induced (classical) and phytohemagglutinin-induced (immune) human interferon were determined. The virus-induced interferon was obtained from leukocytes treated with Sendai virus and from neonatal foreskin fibroblasts treated with Newcastle disease virus. With both classical and immune interferons, the mean response of the trisomic cell lines was three times that of the normal cells, whereas that of the monosomic lines was half or less that of the normal cells, Furthermore, a line trisomic for only the distal half of the long arm of chromosome 21 (q21 leads to qter) also demonstrated increased sensitivity to virus- and phytohemagglutinin-induced interferons, a fact that indicated that the gene responsible for the antiviral effect of interferon, AVG, is located on this part of chromosome 21. Responses to the two categories of interferon (virus-induced and phytohemagglutinin-induced) of individual cell lines of different degrees of sensitivity were strongly correlated (r=0.79). It is concluded, therefore, that despite their physical and antigenic differences, the antiviral expressions of both classical and immune interferons are ultimately mediated by the same genetic locus, AVG.


Advantages and limitations of animal models in the evaluation of antiviral substances. Since many antiviral substances with potential for use in humans are in various phases of evaluation, criteria must be developed for selection of those compoinds with the greatest probability of efficacy and least toxicity. We lack background experience in evaluation of antivirals to permit extrapolation from in vitro tests to use in humans; it is of critical importance, therefore, to develop animal models for evaluation of antiviral substances before trials in humans and to establish guidelines for the relative predictive reliability of in vitro screening and evaluation in animal models. The complexity of drug-host and virus-host interaction and other factors may limit the predictive value of some or all experimental systems. Although the use of animal models is an important phase in the evaluation of antiviral chemotherapeutic agents, the models must be carefully studied and the interaction of drug and virus in the experimental animal specifically defined if optimal guidelines for the predictive value of model systems are to be developed. These guidelines must then be modified as experience is gained with antiviral substances that reach human trials.


Aldosterone and renin-angiotensin responses to stimuli in patients with treated congestive heart failure. Aldosterone responses to physiologic doses of known regulatory factors have been studied in 8 patients with treated congestive heart failure under standard conditions of electrolyte balance and controlled body posture. The response of plasma aldosterone, plasma renin activity (PRA), and in some instances angiotension II, to the brief (120 minute) intravenous administration of ACTH (1.25 mug beta1-24 ACTH in 60 minute and 5 mug in 60 minutes), angiotensin II (30 mug) and potassium (30 mEq.), and to 2 hours in the upright posture, was measured. The plasma aldosterone increase was greatest in response to ACTH (mean increment 20.8 ng. per 100 ml.) followed by that with upright posture (mean rise 17.1 ng. per 100 ml.), potassium (mean 6.7 ng. per 100 ml.) and angiotensin II (5.8 ng. per 100 ml.). PRA rose vigorously following 2 hours of upright posture and fell with angiotensin II infusion, but showed no definite change in response to potassium or ACTH. A diurnal pattern of PRA, angiotensin II, and plasma aldosterone on control days was observed in these patients, with higher prenoon levels falling to a nadir at midnight. The electrolyte and hormone response to administered aldosterone (75 mug over 2 hours) was also studied in the same patients. During aldosterone infusions, plasma aldosterone increased (increment range 36 to 110 ng. per 100 ml.), urine sodium excretion decreased, but no significant change in urine potassium excretion or in PRA was observed. It is concluded that small fluctuations in ACTH secretion, as well as change in body posture, produce marked effects on plasma aldosterone in patients with controlled congestive heart failure. Aldosterone responses to ACTH in these patients is similar to that observed in normal subjects.


The process of cholesterol cholelithiasis induced by diet in the prairie dog: a physicochemical characterization. The rapid induction of cholesterol cholelithiasis in a new experimental model, the prairie dog, has recently been reported by two groups. In this model they were able to induce gallstones in as brief a period as 2 weeks, using a 1.2 per cent cholesterol diet. This unprecedented time intensity or telescoping of the induction process provided a unique opportunity for observation of physicochemical changes occurring rapidly over a short period of time and to correlate these with degree of biliary cholesterol saturation. To make such observations, 97 adult male and female prairie dogs were used in the present study. Seventy-two were fed the high cholesterol diet and sacrificed at intervals over a 14-day period; the remaining 25 were used as controls. The primary objective of this work was to determine whether or not there was any relationship between the in vivo events induced in prairie dog bile and our recently reported detailed observations of cholesterol precipitation phenomena in synthetic bile analogs. In these studies, solutions of physiologically relevant composition were constructed, which, when plotted according to convention on a tri-linear graph, fell within the zone of metastable or suspended supersaturation. These solutions revealed a consistent and previously undescribed liquid crystal to solid crystal phase transition during their approach to equilibrium at 37 degrees C. The in vivo studies of prairie dog bile following rapid induction of supersaturation revealed identical changes. At first, the supersaturated biles were isotropic followed by a period of turbidity and invariable formation of mesophase. After a few days, the liquid crystals decreased and solid cholesterol crystallites appeared. The most striking aspect of these observations is that the in vitro work has now predicted not only static, but dynamic processes as well, with respect to cholesterol precipitation beginning with the metastably supersaturated state and eventuating in gallstone formation. Ancillary new findings, resulting from the high cholesterol diet, included the induction of human-like hyper-beta-lipoproteinemia and rapid hepatocyte degranulation shown by electron microscopy.


Evidence that the platelet retention factor is separate from the factor VIII-related antigen and factor VIII. Plasmas from a pregnant patient with von Willebrand's disease and from a patient with von Willebrand's disease who is taking Premarin had elevated levels of Factor VIII-related antigen and Factor VIII activity without any improvement in their platelet retention abnormality. When these were added to the blood from nonpregnant patients with von Willebrand's disease no significant improvement in platelet retention followed. This suggests that the "retention factor" is independent of the antigenic Factor VIII-like material.