Hepatitis B surface antigen: regional variation in sub-type ratio in the Canadian Red Cross donor population. A total of 344 sera positive for hepatitis B surface antigen from volunteer blood donors at several Canadian Red Cross centres were subtyped for ad and ay specificity by counterelectrophoresis. Of the 50 sera from Toronto 21 (42%) were ad and 29 (58%) were ay; of the 95 from Montreal 82 (86%) were ad and 13 (14%) were ay; of the 199 from Quebec 179 (90%) were ad and only 20 (10%) were ay. The w and r specificities were also determined in 125 of the samples: 123 were w; the 2 samples of r specificity were from Toronto. On the other hand, among 45 sera from patients with acute hepatitis type B in Quebec 13 (29%) were ad and 33 (71%) ay.


Pseudocyanotic pigmentation of the skin induced by amiodarone: a light and electron microscopic study. An unusual bluish discolouration of the nose was noticed in a woman 9 months after she had begun treatment with a coronary vasodilator, amiodarone hydrochloride. Cutaneous biopsies of the nose were obtained 6 and 9 months later for light and electron microscopic studies. In the dermis were histiocytes containing cytoplasmic yellow-brown granules with histochemical properties of melanin and lipofuscin. Ultrastructurally the granules appeared as lysosomal membrane-bound dense bodies similar to lipofuscin. Similar granules were observed at diascopy in both corneas. The pathogenesis is obscure. A storage disease involving the drug or its metabolites cannot be ruled out. Another possibility is that amiodarone accelerates the normal cellular autophagocytosis, resulting in increased production of lipofuscin, which then accumulates in lysosomes because of a deficiency in lipolytic enzymes.


An error in Rh testing in pregnancy.

An error in Rh testing in pregnancy. Anti-D (anti-Rho) in the blood of two Rh-negative pregnant women was believed to be due to active immunization. In the first case, however, antibodies were no longer detectable 2 weeks later. In the second case they disappeared by the end of 31 weeks. It was discovered that both women had been given immune globulin (human) because of exposure to rubella. The globulin given to the first woman probably contained about 0.1 mug of anti-D per ml; that given to the second probably contained about 0.6 mug of anti-D per ml. Both babies were O Rh-positive. Both women were given Rh immune globulin after delivery. Both have completed a further pregnancy and no anti-D has been found on many tests. In tests carried out in 1971 all samples of immune globulin (human) examined contained anti-D, but usually in inconsequential trace amounts.


Virulence of genus Staphylococcus. III. Studies on the pathogenicity to mice. Six representative strains of staphylococci were selected on the basis of DNase activity and inoculated into the mice to study the correlation of the presence of this enzyme to the virulence of the staphylococci. Two strains of staphylococci among them produced DNase but not the free coagulase, and one of these two strains had an obvious virulence against the mice. The results of experiments suggest that there are at least certain strains which may be designated as Staphylococcus aureus among these DNase-producing staphylococci producing no free coagulase. This is an additional evidence that this enzyme activity should be adopted as a criterion to distinguish from the other S. aureus which has lost the free coagulase activity. In this sense, the staphylococci producing neither free coagulase nor DNase are after all Staphylococcus epidermidis, regardless of their ability to ferment  mannitol or not.


Immunological and electrophoretical approaches to macroamylase analysis. The characteristics of abnormally large-size amylase (macroamylase) of the three patients were studied. Amylase isoenzyme patterns of the three macroamylase samples of agar gel electrophoresis were different from the normal one: a post-beta-globulin or middle-ganna-globulin zone was found in macroamylase, while a fast-gamma, and a pre-gamma-globulin or both zones is found in normal amylase. With gel chromatography at pH 3.4, macroamylase dissociated to normalsized amylase. The electrophoretic patterns of the normal-sized amylase were identical with those of normal amylase. The binding proteins of the macroamylase were identified as immunoglobulin IgG in onecase and  immunoglobulin IgA in the other two cases by the immunochemical criteria and immunoenzyme electropherogram. These immunoglobulins bound with serum amylase having normal molecular weight and electophoretic mobility to form macroamylase.


High-molecular-weight hydroxyproline-protein in relation to immunoglobulins M (IgM) in human blood serum. The content of the high-molecular-weight protein-bound hydroxyproline (Hyp) and IgM level in the sera of 30 healthy persons and 29 patients showing changes in gamma and/or beta globulin levels was determined. Statistically significant correlations between quantities of these components have been found. On the base of the present results as well as the results of previous studies authors suggest that the high-molecular-weight Hyp-protein contains immunoglobulin of the IgM class.


Relation of alcoholic liver injury to cirrhosis. The earliest and most reproduceable lesion associated with chronic alcohol abuse is fatty liver. In some alcoholics this may be superseded by alcoholic hepatitis, which may represent the link between the early lesion and cirrhosis. Alcoholic cirrhosis usually begins as a regular, monolobular variety, but is eventually transformed into an irregular, multilobular type. All stages of alcoholic liver injury have now been produced in the baboon, despite high protein and vitamin supplemented diets. Alcohol may therefore now be regarded as a direct hepatotoxin. Epidemiological studies have indicated that alcoholic liver injury begins with an intake of more than 80 g ethanol a day, and that cirrhosis is generally not seen with an intake of less than 160 g per day. The development of cirrhosis correlates with the total duration and amount of alcohol ingested. Complications of alcoholic cirrhosis include iron overload and primary hepatic carcinoma.


Symptoms of Turner's syndrome and interstitial heterochromatin in i(Xq). A girl is presented with "isochromosome" X mosaicism, 45,X/46,X,i(Xq)/47,X,i(Xq),i(Xq). The "isochromosome" had an unusual extra band close to the centromere on one arm. The patient had some signs of Turner's syndrome, myopia and optic nerve atrophy.


Characteristics of ventricular extrasystoles and their prognostic importance: a reappraisal of their method of classification. The concept of two different types of extrasystoles, parasystolic and coupled, depends upon two distinguishing characteristics of these beats. The characteristics of the parasystolic extrasystoles are the invariability of the ectopic cycle together with their independence from the basic rhythm. Coupled extrasystoles demonstrate a dependence upon the basic rhythm although they may express some degree of ectopic variability. The degree of variation of the interectopic interval or its common denominator measures the irregularity of the ectopic parasystolic rhythm. The variation of coupling intervals describes the dependence of the ectopic beat upon the basic rhythm. In a study of 719 electrocardiograms with ventricular extrasystoles, about one-third of the extrasystoles appeared intermediate between these types since they had both variable coupling intervals and variable interectopic intervals. Some of these had total variability of coupling intervals and of the interectopic intervals (random non-parasystolic coupling), and others had limited variation of coupling intervals when expressed in relationship to the total duration of electrical diastole (approximate non-parasystolic coupling). Both these ectopic types appeared to be associated with cardiac disease, and repetitive ventricular extrasystoles. Left bundle branch type extra-with fixed coupling. There were no obvious relationships between the contour and the type of coupling of ventricular extrasystoles. Left bundle branch type extrasystoles with vertical or right axis were the most frequent, particularly in normal subjects, but in the presence of cardiac disease there were more electrocardiograms with right bundle branch type extrasystoles. Extrasystoles in the presence of underlying conduction defects were usually of opposite configuration to this defect. The contour of uniform extrasystoles did not appear to predispose to serious ventricular arrhythmias but multiformity of extrasystoles was an important prognostic indicator. It is suggested that variability of contour and coupling are important signs of inhomogenous conduction and may precede the onset of severe ventricular arrhythmias. Random nonparasystolic coupling and marked multiformity indicate a more sinister arrhythmic state.


[Pacemaker treatment of tachyarrhythmias by programmed rate-related interval stimulation(author's transl)]. Programmed rate-related interval stimulation proved to be a low-risk method when applied to 74 patients with drug-resistant ventricular tachycardia or runs of ventricular extrasystoles as precursors of tachycardia. Atrial flutter was converted to atrial filbrillation and sinus rhythm by atrial interval-related multiple stimulations. Tachycardias, which could not be terminated by simple or double stimulation, were successfully suppressed by sequential ventricular R-R related multiple stimulations. Rare-related stimulation has proved to be an alternative method of treating tachy-arrhythmias unresponsive to conventional means.


[Re-entry mechanism of ventricular tachycardias in the syndrome of nonhomogeneous delayed repolarisation (author's transl)]. The syndrome of Jervell and Lange-Nielsen with its characteristic combination of Q-T-(U) abnormalities and recurrent episodes of ventricular tachycardia (VT) or ventricular fibrillation can serve as a model for establishing recycling excitation (re-entry) as the likely cause of VT. The ECG abnormality in the Q-T-(U) segment indicates the underlying asynchronous repolarisation, probably localized in the His-Purkinje system. Asynchronous delayed repolarisation facilitates re-entry excitations. A personal case is reported which demonstrated two different patterns of inducing premature beats and VT, indicative of two different fascicular pathways of circus movement. The inducing extrasystole determines the circuit pathway and thus the ECG pattern of VT. The VT shows a fixed time relation depending on conduction delay and refractory period, which is a further indication for an underlying re-entry mechanism. Spontaneous changes in time relation or morphology point toward alterations in circuit pathways and can terminate the VT. Clinical findings in three further cases of the syndrome are reported. Furthermore, the possible general significance of these findings is highlighted by the occurrence of the described phenomena in seven patients in whom Q-T-(U) abnormalities exist merely as transient symptomatic disturbances.


Air pollution impact of a suburban highway segment. Design traffic volumes, vehicle emission factors and meterological data are used to assess the impact of a suburban limited-access highway segment. Mesoscale effects are determined by emission inventory techniques. Microscale effects are determined using a pollutant dispersion model developed by the California Division of Highways. The air pollution impact of the highway segment is small, and should not be considered a significant criteria for decisions concerning construction of the highway.


The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Distribution and accessibility to antibodies of some epitopes of the bifunctional enzyme. In the presence of l-threonine, the allosteric effector, most of the antigenic determinants situated in the aspartokinase region of the wild-type enzyme become unavailable to the antibodies raised against a fragment of the enzyme containing this region and devoid of homoserine dehydrogenase activity. The cross-reactivities of the antibodies raised against this fragment (extracted from a nonsense mutant) and a fragment endowed with homoserine dehydrogenase activity but devoid of aspartokinase activity (obtained by limited proteolysis) with the corresponding antigens were studied. The conclusion is drawn that the two fragments, which share an overlapping sequence of molecular weight about 17,000, share at least two antigenic determinants.


The beta-adrenoceptors of the lung mediating inhibition of antigen-induced histamine release. The beta-adrenoceptor stimulants, isoprenaline (IPR), orciprenaline (OPR), terbutaline (TRB), and ITP were studied for effects on antigen-induced release of histamine from guinea-pig lung tissue and for effects on guinea-pig isolated trachea and heart. The order of potency for the agents in the four funct-ons studied were: (a) inhibition of histamine release, IPR greater than OPR approximately equal to TRB greater than ITP equal 0; (b) heart stimulation, chronotropic effect, IPR greater than OPR greater than ITP approximately equal to TRB; (c) heart stimulation, inotropic effect, IPR greater than OPR greater than ITP greater than TRB; (d) trachea relaxation: IPR greater than TRB greater than OPR greater than ITP. These findings suggest that the beta-adrenoceptors mediating inhibition of antigen-induced release of histamine are more related to those mediating trachea relaxation (beta2) than those mediating cardiac stimulation (beta1).


Coagulation, hemostasis, and plasma expanders:a quarter century enigma. Despite more than 2 decades of research, the explanation of the long-known hemostatic failure consequent to the use of some natural and synthetic macromolecular agents as plasma substitutes remains obscure. Conventional clotting parameters are not significantly affected in vivo or in vitro. Dextran, hydroxyethyl starch, and many other colloid macromolecules precipitate Factors I and VIII, fibrin monomer, and perhaps v. W. (von Willebrand) factor(s) from plasma, rendering at least the first three insoluble, in relation to the molecule size and concentration of the colloid, and for dextran, its intrinsic viscosity. The precipitate, rich in Factors VIII and I, redissolves on warming, and reprecipitates on cooling, behaving as a cryo-Factor I. In composition it closely resembles the cryoprecipitate obtained by slow-thawing of plasma. Both clot faster with thrombin than the parent plasma. The amount precipitated from plasma by dextran or hydroxyethyl starch varies very widely from individual to individual. Cryo- of dextran-precipitable material can be obtained by interacting purified Factor I with a miniscule amount of thrombin. Dextran, hydroxyethyl starch, polyvinyl pyrrolidone, some forms of gelatin, and several polyamino acids accelerate thrombin clotting of normal plasma, several dysfibrinogenemic plasmas, or Factor I. Albumin, hemoglobin, some modified gelatins do not. Poor platelet thromboplastic function appears some hours after dextran infusion, associated with morphologic capillary abnormalities that strikingly resemble those in v. W. disease. We postulate that the hemostatic defect associated with the use of plasma substitutes is a form of induced v. W. disease or disseminated intravascular clotting, ensuing from precipitation and removal of v. W. factor(s), Factors VIII and I, microcirculatory abnormality, and platelet malfunction. The latter two supervene some time after administration of dextran. It reported antithrombotic activity is perhaps referable to the same action.


Partial purification and identification of a reversible decapacitation factor from rabbit seminal plasma. Efforts have been directed toward partial purification and identification of a reversible decapacitation factor from rabbit seminal plasma. Five fractions were consistently obtained following fractionation of rabbit seminal plasma on a Sephadex G-200 column. Only the first two fractions had decapacitation factor (DF) activity. On SDS polyacrylamide gel electrophoresis, several carbohydrate-and protein-containing components were found in each fraction from the Sephadex column. Further, DF activity could be obtained in the precipitate resulting from cetyl pyridinium chloride (CPC) treatment of the active fractions from the Sephadex G-200 column. In the comparison of the components observed with SDS gel electrophoresis throughout these fractionation procedures, the reversible decapacitation activity appeared to be associated with a component containing protein and carbohydrate. From these studies, then, reversible decapacitation activity of rabbit seminal plasma appears to be associated with a glycoprotein of an approximate molecular weight of 115,000.


Recovery of spermatozoa from human vaginal washings. Sperm survival, in terms of numbers, motility, viability, and metabolic activity, was studied in vaginal washings obtained daily from 22 healthy, fertile, married women during one complete menstrual cycle. The numbers of sperm (counted on Papanicolaou-stained filters) were never large compared to the number of sperm in a single ejaculate. Forty-eight hours after intercourse, only 6% of the specimens showed any evidence of sperm. Motile sperm were observed in only six of 94 postcoital specimens examined within 12 hours after intercourse. Lack of sperm in specimens collected after reported intercourse was associated with contraceptive practices other than the rhythm method, which most of the subjects were using. Fluorochromatic studies of recovered sperm treated with acridine orange indicated sperm viability and phase contrast studies of sperm treated with tetrazolium salts revealed metabolic activity. Our data on sperm motility, numbers, and staining reactions support the conclusion that the small number of sperm that remain in the vagina after intercourse quickly become inactivated.


Antihistamine blockade of the ovarian hyperstimulation syndrome. II. Possible role of antigen-antibody complexes in the pathogenesis of the syndrome. Antihistamine can prevent the ovarian hyperstimulation syndrome in the rabbit. The mechanism underlying this experimental observation has not been elucidated. Our experiments were directed at the question of whether antigen-antibody complexes are a factor in the development of the ovarian hyperstimulation syndrome. The results do not support the theory that antigen-antibody complexes play a role in the pathogenesis of this syndrome.


Cellular proliferation and renewal in the various zones of the hamster epididymis after colchicine administration. Cellular proliferation was examined in the various regions of the excurrent ducts of the reproductive system of the male golden hamster after colchicine administration. Each of the two zones of the ductuli efferentes displayed only infrequent mitotic figures. Similarly, the basal cell population of the epididymis as well as the epithelium of the vas deferens exhibited too few mitotic figures to allow meaningful evaluation of cellular proliferation. The following observations were reported. 1. The principal cells of the entire epididymis are characterized by a singular diurnal cycle of proliferative activity reaching its maximum about 3 P.M. and its nadir about 9 A.M. 2. A circadian rhythm of cell division is also found in the principal cells of each of the six zones of the epididymis, although the time of maximal activity may vary from zone to zone. 3. The most actively proliferating region of the excurrent duct system is zone 3 of the epididymis, whereas the least active region is the ductuli efferentes. 4. The renewal rates of the principal cells vary considerably at various points along the excurrent duct system, ranging from cells which renew themselves as many as 10 times to cells which fail to renew themselves at all during the normal life-span of the animal.


Testis antigens of man and some other primates. Rabbit antisera raised aginst testis preparations of human, chimpanzee, rhesus monkey, and baboon origin were used to study testis-specific antigens within and among the four primate species. Antisera were absorbed with serum, liver, kidney, and spleen preparations of the respective species against which they had been produced. Immunoelectrophoretic analysis of testis extracts, using the absorbed antisera, indicated the following minimum numbers of testis-specific antigens for each species: man, 10; chimpanzee, 8; rhesus monkey, 10; and baboon, 8. Most of the testis antigens were cross-reactive among species. The results suggest that human spermatozoa possess at least four to five specific antigens which originate in the testis. The antigen that induces sperm-immobilizing antibody cross-reacted among the four species of primates. Human and rhesus monkey sperm reacted equally in the immobilization system. Testis extracts from each primate species were capable of removing the sperm-immobilizing activity of human immune sera by absorption. Testis proteinase activity, as determined by using a gelatin membrane substrate, was inhibited by the gamma-glogulin fractions of rabbit and rhesus monkey antisera and also appeared to be cross-reactive among the species.


Melatonin effects on brain. Interaction with microtubule protein, inhibition of fast axoplasmic flow and induction of crystaloid and tubular formations in the hypothalamus. [3-H]Melatonin administered in vivo in the rat cisterna magna became associated with a vinblastine-precipitable protein. Melatonin treatment decreased microtubule protein content by 44% in the arcuate-median eminence region and by 19% in the remaining hypothalamic block, being without significant effect on the cerebral cortex. Superior cervical gangliectomy but not pinealectomy increased microtubule protein content of the rat hypothalamus. Norepinephrine brought about a significantly greater decrease in hypothalamic microtubule protein levels of ganglionectomized rats than in sham-operated or in ganglionectomized-pinealectomized animals. Melatonin treatment induced in most of the axons ending in the pericapillary zone of the rat median eminence crystaloid and tubular formations. Rapid axonal transport in retinal ganglion cells of rabbits was inhibited to the extent of 71.9 and 87.2% by previous exposure to 1.5 of 15 mu g of melatonin intravitreally; melatonin did not affect retinal protein synthesis in this experimental model. These results suggest that melatonin interacts significantly with microtubule or actin-like protein in brain.


Steroid-binding proteins in rabbit plasma: Separation of testosterone-binding globulin (TeBG) from corticosteroid-binding globulin (CBG), preliminary characterization of TeBG, and changes in TeBG concentration during sexual maturation. Two distinct steroid-binding proteins are present in rabbit plasma. One of the proteins (TeBG) binds [3-H]5 alpha-dihydrotestosterone (5 alpha DHT) and [3-H]testosterone. The affinity of this binding protein for 5 alpha DHT was 3-4 times greater than for testosterone. Binding of [3-H]5 alphaDHT could be inhibited by unlabeled 5 alpha DHT, testosterone, 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), and 17 alpha-methyl- B-testosterone (skf) 7690). The relative affinity of the competitors was: 5 alpha DHT greater than 3 alpha-diol greater than testosterone greater than SKF 7690. The antiandrogens, cyproterone (1,2 alpha-methylene-6-chloro-pregn-4,6-diene-17 alpla-ol-3,20 dione), cyproterone-17-acetate, and 6 alpha-bromo-17 beta-hydroxy-17 alpha-methyl-4-oxa-5 alpha-androstan-3-ine (BOMT) were ineffective in competing for [3-H5d alpha DHT binding sites, as were 4-androstene-3, 17-dione, 17 beta-estradiol (E2), progesterone, and cortisol. The formation of the [3-H]5 alpha DHT-TeBG complex was extremely rapid; the binding reaction was essentially completed in 15 s. The complex dissociated rapidly in the presence of charcoal. The dissociation rate constant (Kdiss) was 0.157 min- minus 1 and the dissociation half-time t-1/2) was 4.5 min. In the presence of charcoal and unlabeled 5 alpha DHT the Kdiss was 0.268 min- minus 1 and the t=1/2 was 2.6 min. The sedimentation coefficient of TeBG was congruent to 4.6 S and its molecular weight, estimated by gel filtration on a calibrated Sephadex G-200 column, was congruent to 75,000. The concentration of TeBG in male rabbit plasma decreased with sexual maturation and was approximately three times higher in adult females than in adult males. The other protein (CBG) bound both [3-H]cortisol and [3-H]progesterone. Binding of these compounds could be inhibited by unlabeled cortisol and progesterone, but not by unlabeled 5 alpha DHT, testosterone, or E2. CBG had a sedimentation of congruent to 3.9 S and an apparent molecular weight of congruent to 105,000. TeBG could be separated from CBG by a 60% ammonium sulfate precipitation and by gel filtration chromatography. Both proteins are thermolabile; TeBG is inactivated at temperatures above 30 degrees C and CBG is inactivated at temperatures above 50 degrees C.


Immunologic and virologic aspects of secretory immune system in human respiratory tract. Many external mucosal surfaces in man are replete with immunoglobulin containing plasma cells and thymic dependent (T) lymphocytes. Immunization with viral vaccines administered via different routes have indicated that viral specific secretory immunoglobulins particularly of gamma A class are synthesized locally in the external mucosal surfaces. Local availability of viral antigens especially after local mucosal immunization appears to be the most effective means of inducing viral specific secretory antibody responses in the respiratory tract. Similarly, local induction of specific cellular immune responses in the respiratory tract and tonsilar lymphoid cells has been demonstrated after intranasal immunization with viral vaccines. Generally, the locally induced secretory antibody and cell-mediated immune responses in the respiratory tract appear to be independent of the immune response in the systemic sites, with little or no contribution from circulating immunoglobulins and T-cells. Information obtained after natural or vaccine induced infections with polio, influenza, measles, rubella and other viruses suggest that the outcome of a reinfection challenge in the respiratory and alimentary tracts is determined to a major extent by the presence and level of pre-existing secretory antibody. Although the precise role of locally induced cellular immunity in protection against viral infection remains to be determined, these observations suggest that the mechanism of immunologic defence in external surfaces may be mediated largely through specific secretory immunoglobulin and cellular immune response.


Immunity to influenza.

Immunity to influenza. Immunity to influenza virus may be considered from the standpoints of viral and hostfactors. Amonst viral factors the phenomena of antigenic 'shift' and 'drift' of the surface antigens. Hemagglutinin HA and neuraminidase NA, are of utmost importance in enabling the virus to combat host immunity and to produce recurrent pandemics and epidemics of disease. 'Shift' involves major changes in the antigenic character of the HA and NA antigens, and serological studies reveal little or no crossreactions between antigens of different subtypes. However, immunological 'memory' may exist between the surface antigens of viruses of different subtypes, for example between the hemagglutinins of Asian (H2) and Hong Kong (H3) subtypes. Antigenic 'drift' occurs more frequently than 'shift' and involves subtle changes in the antigenic configuration of HA and NA within a subtype. It is clear that the hemagglutinin antigen contains a multiplicity of antigenic determinants, one or more of which remain stable (CR determinants) whilst others (strain-specific determinants) change completely during antigenic 'drift'. Amongst host factors humoral antibody to HA and NA appears to be a reasonable index of natural or vaccine induced immunity to infection. As far as the specificity of such antibodies is concerned, it is likely that future studies will place considerable emphasis on the distinction between antibody for the stable (CR) antigenic determinants and antibody to strain-specific determinants. It seems likely that antibody to strain-specific determinants is of more relevance to immunity than CR antibody. The studies of Virelizier et al. described elsewhere in this Symposium employing single-radial-diffusion tests give hope of simple methods for the investigation of such antibody specificity. There is no evidence that antibody to the internal, antigenically stable ribonucleoprotein is related to immunitywhilst the role of the other internal antigen, the membrane protein, in immunity is at present unknown. Numerous studies have been carried out to establish the importance of secretory antibody in the respiratory tract. Although secretory IgA specific for the influenza virus has been clearly demonstrated in the respiratory tract, the levels are low and its significance is not yet established. Whilst the role of cellular aspects of immunity to influenza in man remains to be established there is no doubt from the recent studies of Tyrrell and his colleagues that delayed hypersensitivity to influenza antigens can be demonstrated. In considering the host aspects of immunity it is important to attempt to distinguish factors which are merely an index of past exposure and those which are involved with the actual mechanism of immunity. It is likely that animal experimental models, in particular with inbred mice, will provide an important contribution in this field of investigation.


Myxoviruses.

Myxoviruses. Myxoviruses can be divided into 2 groups, orthomyxoviruses and paramyxoviruses. the former comprise the influenza group which is subdivided into types A,B and C. Influenza B and C are purely human pathogens but influenza A, which includes a large number of antigenic subtypes, occurs in nature in pigs, horses, birds and man. All influenza A viruses irrespective of origin are chemically, biologically and genetically related. The epidemics which they cause are curious and puzzling and are an important cause of morbidity. Paramyxoviruses consist of human and animal parainfluenza viruses. The former comprise parainfluenza types 1,2,3 and 4 and mumps viruses, and, rather less certainly, measles and respiratory syncytial virus. The main animal paramyxoviruses are parainfluenza 3 (agent of shipping fever) in cattle; NDV (cause of fowl pest) and Yucaipavirus in birds; Sendai and PVM in mice; Nariva virus in rodents; possibly bovinerespiratory syncytial virus; and SV5 and SV41 in monkeys. The viruses and their place in nature will be reviewed.


Studies on parainfluenza-3 virus neuraminidase. Various parainfluenza-3 virus (PIV-3) strains differ in neuraminidase (NA) activity, some being neuraminidase strong (NAS) and some neuraminidase weak (NAW). No difference in the ability of these strains to elicit antibody activity to hemagglutinin (HA) or NA of PIV-3 was observed. Samples of formalin-treated PIV-3 released more N-acteylneuraminic acid (NANA) than samples of Piv-3 not treated with formalin when these samples were incubated with the substrate sialolactose for 16 h at 37 degrees C. The initial activity of PIV-3 NA, and the Km-value, were similar for a NAS strain whether it was treated with formalin or not. The results suggest that formalin stabilized the NA structure of PIV-3. A NAS strain of PIV-3 dialyzed against distilled water showed an increased initial neuraminidase activity as compared to virus dialyzed against isotonic NaCL or to non-dialyzed virus. The increased enzyme activity indicates an increased affinity of NA tothe substrate sialolactose as an increased Km-value was also obtained. It is suggested that PIV-3 neuraminidase facilitates the penetration of the virion through the mucociliary barrier of the respiratory tract by releasing the virus from inhibitors in the gel phase. It has been found that antibody in the gel phase of nasal secretion inhibits the NA activity of PIV-3 Following exposure of a gel phase of bovine tracheal secretion to a NAS strain of PIV-3 an increased initial viscosityof this phase was observed. The gel phase of bovine nasal secretion showed a structural change in scanning electronmicroscopy following exposure to a NAS strain. Stimultaneously an increased amount of free N-acetylneuraminic acid was found in the gel.


Plasma proteins in canine gastric lymph. Because it has not been possible to collect gastric lymph by cannulation of lymph vessels, the methods of renal micropuncture were adapted to collection and analysis of 0.5- to 3-mu samples obtained by puncturing lymph vessels on or immediately adjacent to the ventral surface of the dog's stomach. Samples of gastric lymph collected immediately after completion of surgical exposure of the stomach were subjected to acrylamide gel electrophoresis, and they were found to show all the protein bands exhibited by simultaneously collected samples of plasma. Quantitative analysis of similar samples by cellulose acetate electrophoresis gave the following ratios of protein concentration in gastric lymph to plasma: total protein, 0.51; albumin, 0.68; total globulins, 0.42; alpha1-globulin, 0.49; alpha2-globulin, 0.62; beta-globulin, 0.35; fibrinogen, 0.39; and gamma-globulin, 0.46. When samples were collected over 4 hr the concentrations of all proteins in lymph increased, and an argument is presented that this was not the result of increasing small pore size as the result of injury. When the mucosa was irrigated with solutions of dithiothreitol, a substance causing plasma shedding, the concentrations of proteins in gastric lymph over a 4-hr period were similar to those found in control experiments. Gastric lymph mixed with 100 mN HCl was found to have proteolytic activity.


Relationship of hepatitis B antigen in cirrhosis and hepatoma in Thailand. An etiological significance. A study of 221 patients revealed that detectable hepatitis B surface antigen (HBS Ag) was found in 16.3% of 49 patients who had hepatoma associated with cirrhosis. None of the 8 hepatoma patients without cirrhosis had detectable HBS Ag in the serum. When known causes of cirrhosis were excluded, HBS Ag was present in 18% of 22 patients. Positive alpha-1-fetoprotein (AFP) was found in 25 of 49 cases (51%) of hepatoma with cirrhosis but was found only in 1 of 8 cases (12.5%) of hepatoma without cirrhosis. Of 25 patients whose AFP was positive, HBS Ag was also present in 7. The latter was detected in only 1 of 24 patients in whom AFP was not detected. This study suggests that HBS Ag is closely associated with hepatomas in cirrhotic patients but not in noncirrhotic patients with hepatoma.


The effects of fast neutrons on inoperable carcinoma of the stomach. Thirty-nine unselected patients suffering from inoperable, recurrent, or residual adenocarcinoma of the stomach were referred for palliation with fast neutrons from the Medical Research Council's cyclotron at Hammersmith Hospital. A full course of 1440 rads given in 12 treatments over 26 days was administered to the patients. Because of the relatively low energy (7-5 MeV) of the beam from this particular machine, it was not possible to deliver the full dose uniformly throughout the tumour except in extremely thin patients. Pain, dysphagia, vomiting, and bleeding were relieved in the majority of cases. The side effects were minimal and easily controlled. Palpable masses disappeared. Five patients required surgery after neutron therapy. All the incisions were made through irradioated tissue and all except one healed normally. Tumour was present outside the treated area, but the absence of any palpable mass within the treated area was a consistent finding. Radiologically, the stomachs remained abnormal and later changes included gross mucosal abnormality and shrinkage. Fourteen patients came to necropsy and in 10 no tumour was present macroscopocally. Tumour cells were seen in all except two cases but these were few, surrounded by dense fibrous tissue, and may not have been viable. The remaining stomach was abnormal with a thickened wall and destruction of mucosa. Three of the four cases in which macroscopic tumour was present received less than the standard dose because of the inadequate penetration of the beam. Excellent regression of tumors was achieved by the neutrons, but the stomachs did not recover from this satisfactorily. Gastrectomy four to six months after treatment is therefore suggested. This operation and other surgical procedures in other patients were successfully carried out. There is a need for higher energy neutrons to improve treatment and extend it to patients of thick-set build.


Structural studies of the Xenopus 19S immunoglobulin and 7S immunoglobulin and two immunoglobulin-like proteins. Xenopus laevis 19S and 7S immunoglobulins (Ig) were extensively reduced and alkylated, their H and L chains spearated and their molecular weights determined. Two kinds of L chains of molecular weight 25,000 and 27,000 were revealed by SDS-polyacrylamide gel electrophoresis. In addition two Ig-like proteins consisting of heavy chains only, of 19S H-type and with similar molecular weight, were detected in Xenopus serum ans isolated. These proteins share common antigenic determinants with Xenopus 19S Ig heavy chains and are devoid of light chain determinants.


Humoral and cellular aspects of immunologlobulin allotype suppression in the rabbit. III. Production of anti-allotypic antibody by suppressed animals. Rabbits heterozygous at the 'b' locus (b4b6 or b5b6) were suppressed for b6 production. These suppressed rabbits were immunized to produce anti-allotypic antibody against: (a) determinatns not possessed in their genotype; (b) the suppressed b6 determinants.


Blocking antigen-antibody complexes on the T-lymphocyte activation during in vitro incubation before adoptive transfer. Following appropriate immunization of mice with CGG or DNP-CGG spleen cells release antigen upon incubation in vitro. The release can be detected either by 'self-stimulation' of the primed cell population, so that upon adoptive transfer antibodies to CGG and DNP are produced without need for further administration of antigen, or by stimulation of indicator (primed) cells. Boosting mice with antigen in saline following initial immunization with antigen in adjuvant proves the most effective method of loading spleen cells with antigen. A small fraction (10- minus 4- minus 10- minus 5) of the antigen used for boost is retained. The antigen appears to be held on the cell surfaces in the form of antigen-antibody complexes, for it is retained on the cells for up to 13 weeks in the presence of circulating antibody, during which time its capacity to self-stimulate can be inhibited by host serum in  vitro. Activated thymus cells take up detectable amounts of antigen-antibody complexes. Complexes obtained by immunization with antigen in adjuvant can be inactivated by trypsinizing spleen cells. Trypsinization of separated T and B cells reveals detectable amounts of complex on the T cells, Complexes detected on T cells in this way are believed to be equivalent to the blocking complexes operative in transplantation and tumour immunity.


Blocking antigen-antibody complexes on the T-lymphocye surface identified with defined protein antigens. II. Lymphocyte activation during the in vitro response. Antibody-forming cells against DNP (DNP-AFC) appear in nonantigen-stimulated cultures of spleen cells from mice which were boosted with antigen DNP-CGG or TNP-KLH in saline several weeks before the vitro cultures were started. The similar kinetics of the appearance of DNP-AFC in antigen-stimulated and non-antigen-stimulated cultures suggests that new B cells were triggered during the in vitro culture. The data supporting the concept of antigen being retained in the body for long periods of time in the form of antigen-antibody complexes on the T-cell surface is discussed.


Carrier and hapten functions in immune deviation. II. Role of hapten and carrier on the helper functions of the T cells. The kinetics of haemagglutinating and haemolytic antibody synthesis to the hapten and to the carrier determinants were studied in guinea-pigs injected  intravenously with large doses of the carrier, or of the hapten conjugated to an homogous or heterologous protein carrier and subsquently immunized with the hapten-carrier conjugate in Freund's complete adjuvant. The animals treated with the heterologous conjugates exhibited enhanced reactions to the hapten and supressed reactions to the carrier, whereas the animals injected with the homologous conjugate showed depressed reactions to the hapten but unaffected reactions to the carrier. Pretreatment with the carrier alone-seemed to have no effect. These experiments confirm that the hapten determinant must act at the T-cell level but do not exclude the possibility that it could also act at the B-cell level. On the other hand, they allow the dissociation of the different components of the immune response directed against the hapten and carrier determinants of the antigen molecule in immune deviation.


Demonstration of a unique antigenic specificity for the collagen alpha1 (II) chain from cartilaginous tissue. The rabbit antibody response to native collagen (chain composition [alpha1(II)3) from cartilaginous tissue, has been examined by agglutination assays, gel diffusion, haemagglutination-inhibition studies, and immunoadsorption. The results show that the rabbit antibody response to the cartilage-type collagen is characterized by considerable reactivity to both helical [alpha1(II)]3 as well as alpha1(II) chains. This is in contrast to the rat antibody response to the same antigens where titres are generated to largely helical antigenic determinants. Similarly to the rat response, rabbit antibodies to [alpha(II)]3 exhibit no strong cross-reaction with the genetically distinct [alpha(I)]2ALPHA2 collagen or its component chains. Strong cross-reactions were, however, observed between bovine and chick alpha1(II) chains. One of the major antigenic sites on [alpha1(II)]3 collagen appears to reside in the sequence represented by CB-11, a peptide derived from the helical portion of the [alpha1(II)]3 molecule after cyanogen bromide cleavage. The data, however, are compatible with the presence of other antigenic determinants which are probably located in the amino- and carbocy-terminal portions of the molecule.


Antigens associated with chemically induced intestinal carcinomas of rats. Rabbits were immunized with extracts of primary or grafted intestinal adeno-carcinomas induced by carcinogenic drugs in inbred rats. After absorption with normal tissue extracts, the antisera were able to recognize three tumor-associated antigens. Two of them were glycoproteins, present in cancer cells but also, in trace amounts, in mucous cells of the normal digestive tract. The third antigen is not detectable in the normal digestive system, but present in normal spleen; on im-unofluorescence, it is not located in the cancer cells, but in polymorphonuclear cells infiltrating the tumor. None of the three antigens cross-reacts with human carcinoembryonic antigen, or human or rat alphafetoprotein. On the other hand, one of the glycoprotein antigens is immunologically related to the human blood group A substance.


Cell-mediated immunity to moloney sarcoma virus in mice. I. Analysis of antigens responsible for lymphocyte stimulation in regressor mice. Purified viruses, viral antigens, and cell extracts have been tested for their ability to stimulate protein synthesis by MSV-M-1 regressor spleen cells from BALB/c mice. Immune, but not normal cells, responded to MSV-M, but not to MSV-G virus-1, and to the type-specific viral envelope glycoprotein from MSV-M virus. Extracts of mouse embryo fibroblasts transformed by either MSV-M or MSV-G, however, specifically stimulated MSV-M regressor spleen cells. Cells stimulated by different antigens, and by phytohaemagglutinin, had the same sedimentation profile and were identified as T-lymphocytes.


Epstein-Barr virus-induced transformation of human leukocytes after cell fractionation. The efficiency of transformation of human lymphocytes after infection with Epstein-Barr virus (EBV) was determined in fractionated and non-fractionated preparations derived from 16 human cord blood samples and two blood samples from adult donors. The transformation efficiency of macrophage-depleted leukocytes was consistently lower as compared to non-fractionated leukocytes. Additional depletion of B-cells resulted in a further decrease. Reduction of T-cells, however, did not influence significantly the transformation rate. In non-fractionated leukocyte cultures, as well as in macrophage-depleted and B-cell enriched cultures, colonies of transformed cells were regularly observed within the first week of cultivation. All cell lines established after EBV-infection revealed membrane-bound immunoglobulin. Reconstruction of macrophages-depleted, B-cell enriched or B-cell depleted cultures with autologous macrophages resulted in an increase of the transfromation efficiency up to the values of non-fractionated leukocyte preparations. Addition of heterologous human embryonic lung fibroblasts resulted in a similar increase. The results support the interpretation that EBV transforms only those cells of the hematopoetic system which are derived from the bone-marrow entity. The transformation efficiency is considerably increased by co-cultivation of lymphocytes with macrophages and heterologous human fibroblasts which seem to excert a feeder-layer effect by enhancing survival of lymphocytes in vitro.


Refractoriness of lymph-node cells from tumour-bearing animals. In vitro lymphocyte stimulation by mitomycin-C-blocked tumor cells has been used to demonstrate tumor-specific antigens in syngeneic murine systems and to follow the evolution of tumor immunity with the tumor-bearing state. Mitomycin-blocked tumor cells stimulated syngeneic lymphocytes from normal mice, from those bearing small tumors (less than 1 cm in diameter) and from tumor-immune mice, sensitized by tumor-cell inoculation and subsequent tumor removal, to undergo increased DNA synthesis as measured by the incorporation of tritiated thymidine. However, lymph-node cells from mice bearing tumors over 1 cm in diameter appeared to be maximally stimulated in vivo and incapable of further stimulation by the same tumor cells in vitro. This was reflected by the progressively increasing background levels of nucleic acid synthesis with the length of tumor-bearing and the size of the tumor. Although lymph-node cells from mice with large tumors did not respond to the same tumor cells in vitro, they did have normal responses to PHA. Within 7-14 days of surgical removal of the tumor, specific lymphocyte responsiveness and background activity returned to previous normal levels, but reinoculation with 10-6 tumor cells resulted in progressive tumor growth and loss of specific  in vitro responsiveness when the second tumor had reached the critical size of 1 cm in diameter. Brief exposure of tumor-immune lymph-node cells to a soluble antigen extract of the same tumor resulted in a marked increase in DNA synthetic activity compared to that obtained after exposure to a different tumor extract, muscle extract or medium alone underwent stimulation when cultured with mitomycin-blocked tumor cells. However, normally responsive tumor-immune lymph-node cells, after brief exposure to a soluble antigen extract of the same tumor, initially underwent increased DNA synthesis, but were incapable of further stimulation by mitomycin-blocked tumor cells. Tumor antigen, alone or complexed with antibody, was also demonstrated in the sera of mice bearing large tumors and is thought to be responsible for the refractoriness of lymph-node cells from these mice to further stimulation in vitro. These experiments demonstrate that tumor size and the consequent antigen load to which the tumor-bearing animals is subjected have a profound effect on tumor-specific lymphocyte responsiveness.


Isolation of type-C viruses from the Asian feral mouse Mus musculus molossinus. N-tropic and xenotropic type-C viruses have been isolated from the wild Asian mouse subspecies M. musculus molossinus. By host range, morphologic and some immunologic criteria these viruses appear closely related to the previously studied murine type-C viruses isolated from highly inbred laboratory strains of mice.


Immunological mechanisms in the pathogenesis of virus-induced murine leukemia. II. Characterization of autoreactive thymocytes. The nature of the reaction and the type of effector cells involved in the reactivity of thymocytes from Moloney murine leukemia virus (MuLV-M)-carrier mice against normal syngeneic target cells has been further characterized. The reaction is mediated by viable MuLV-infected thymocytes. Lysates of carrier thymocytes are not more effective in causing target cell reduction than are lysates of normal thymus cells. Soluble mediators do not appear to be involved in the reaction. The thymocytes mediating the reaction are non-adherent cells positive for the theta-C3H and H-2-k alloantigens, but are negative for detectable murine IgG determinants. Their resistance to corticosteroid treatment further identifies them as functionally mature immunocytes.


Quantitative estimation of mouse mammary tumor virus (MTV) antigens by radioimmunoassay;. A radioimmunoassay for the antigens of the mouse mammary tumour virus has been developed. -125Iodine-labelling of intact virus [derived from mammary tumours of (C3H TIMES 020)F1 mice] followed by ether extraction and separation of the ether and water layers resulted in a separation of the labelled material into two main groups of antigenic components. The intact labelled material from each group was separated from viral debris and other possible contaminants by affinity chromatography. The antigens of the ether phase were proved to belong mainly to the viral nucleoid whereas the water phase contained mainly envelope antigens. No type-specific antigens could be demonstrated in either of the phases. Radioimmunological assessment of plasma revealed that in the plasma of MTV-S- and MTV-P-positive animals viral antigens were only measurable when palpable mammary tumours were present, whereas in MTV-L-positive tumour-bearing animals some negative samples were found. In milk of individual nontumour-bearing mice a close correlation was found between the expression of viral antigens and the generally accepted presence of virus in the strain of mice. In milk, viral antigen levels tend to increase with parity with a possible decrease after a finite number of pregnancies. In a pilot study the presence of MTV antigens could also be demonstrated in epididymis extracts of male GRS/A mice. Genetical analysis of the low-virulent MTV-L by radioimmunoassay of milk is in progress.


Expression of virus-associated antigens and immune cell functions during spontaneous regression of the Friend viral murine leukemia. Spontaneous regression and/or remission of Friend virus (FV)-induced splenic erythroblastic leukemia was observed in CD-1 mice infected with several isolates of FV. Regression of splenic tumor was accompanied by the loss of both specific FV-induced cell membrane antigen (FVMA) and virus group-specific antigens (gsa) from the spleen cells. The frequency (percentage) of immunoglobulin-positive cells (B) and thetapositive cells (T) in the spleen was markedly decreased during leukemia progression, but there was a subsequent increase during regression. The appearance of gsa-positive (gsa+) cells in peripheral blood correlated well with the early progressive and regressive phase of leukemia (up to 7 weeks after infection). Later, the presence of these cells became unpredictable in regard to status of disease. Gsa+ blood cells reappeared in most mice with regressed splenic tumors, suggesting persistence of the virus complex in the animals. Antibody responsiveness as determined by the numbers of hemolytic plaque-forming cells, PFC, after a single immunization with sheep red blood cells (SRBC), was suppressed in leukemia progression and recovered, spontaneously, during regression of leukemia. However, hemolytic PFC elicited by antigen in both progressors and regressors expressed the specific virus-induced membrane antigen, FVMA, detectable by the PFC-inhibition test with specific antiserum and complement. Recovery of immunological responsiveness also included the spontaneous appearance of virus-neutralizing antibody to FV. However, this was not paralleled by the appearance of antibody to FVMA. Traces of anti-FVMA antibody activity were occasionally detectable in serum of both progressors and regressors and did not correlate with virus neutralization, in individual mice; This may explain the susceptibility of regressors to secondary relapse and to reinfection.


Outpatient vs hospital methadone detoxification: an experimental comparison. This study compares the effectiveness and cost of hospital narcotic detoxificationand outpatient narcotic detoxification using methadone. Forty heroin addicts seeking narcotic withdrawal were randomly assigned to treatment groups. Methadone treatment was planned for no longer than 10 days while psychiatric supportive services were made available to continue as needed. The results suggest little benefit from either treatment approach and there was no significant differences between the treatments. Only a few patients achieved a drug-free state, and of those who did, all but one had returned to use of narcotics within 2 months. The data indicate that detoxification using methadone is a palliative procedure. The cost of outpatient detoxification can be accomplished for approximately one-tenth the cost of inhospital treatment without significantlyaltering the effectiveness of treatment.


Histochemical conditions influencing metachromatic staining. A comparative study by means of a model system of polyacrylamide films. The influence of different histochemical conditions on some metachromatic staining reactions has been studied using polyacrylamide films containing pure glycostaminoglycans. The films were incubated in fixatives without staining, and in glycerol, diethylene glycol and other glycols, formamide, N,N-dimethylformamide, dimethyl sulphoxide and ethanol (of several concentrations) after staining and their absorption (metachromatic) spectra recorded. In the case of heparin and heparan sulphate the metachromasy was disturbed when the films were immersed before staining in some fixative solutions containing formaldehyde and acid. After equilibration of stained films in organic solvents, changes in the absorption peaks were found to depend on the type and concentration of solvent, the type of glycosaminoglycan and the type of dye. Films containing glycosaminoglycan plus protein were used to investigate the blocking of the metachromatic reaction as the result of ionic interactions with proteins. The parameters that influence this phenomenon (e.g type of protein, glycosaminoglycan and dye, pH of staining) are discussed and a three-dimensional picture is introduced which can explain some of the results obtained in these experiments.


Histochemical observations of the haemocytes of Locusta migratoria. Five of the six categories of haemocytes of Locusta migratoria, that is, the plasmatocytes, spherule cells, granulocytes, coagulocytes and oenocytoids, contain conspicuous granules of mucosubstance in their cytoplasm. The mucosubstance has been characterized by using a series of histochemical tests, including Alcian Blue staining at different pH levels and salt concentrations, the periodic acid-Schiff (PAS) test, the high iron diamine test, enzymatic digestions and sequential staining methods. The results indicate that four different mucosubstances occur in a granular form, although not all four are found in every blood cell type. The mucosubstances are a neutral glycoprotein and neuraminidase-resistant, sulphated and non-sulphated sialomucins. The non-sulphated sialomucin occurs in both periodate-reactive and -unreactive forms.


Glycosaminoglycans of hair follicles of dog skin. Alcian Blue staining in MgCl-2 of various concentrations revealed that the basement membrane of dog hair follicles contains a large amount of glycosaminoglycan that increases with age, varies with breed, and is significantly greater than that of dermal collagen. This material is highly sulphated and of low molecular weight. Glycoprotein is also present in significantly greater amount than in dermal collagen. Active hair matrix contains clycosaminoglycan in similar amounts to epidermis but the glycoprotein content is much greater and staining is abolished in the keratinized cortex.


Staining of gastrin cells with lead-Haematoxylin. Fixation of fragments of human antral mucosa with Helly's fluid allows gastrin-containing cells to be identified by an immunofluorescence technique. Lead-Haematoxylin staining carried out on the same sections shows selective reactivity of the immunofluorescent cells. These findings support the identification of gastrin cells with G cells, known from previous studies to react with lead-Haematoxylin.


The lysosomal permeability test modified for toxicity testing with cultured heart endothelioid cells. A modified lysosomal fragility test is described which is suitable for use with cultured cells. The permeability (fragility) of the lysosomal membranes of the cells to the substrate beta-glycerophosphate is measured by assessing the degree of particulate lysosomal straining seen after exposing the cells to the Gomori acid phosphatase staining reaction under carefully controlled conditions. Monolayer cultures of endothelioid cells from the hearts of neonatal rats have been used in all experiments. The time-course of lysosomal straining for cells exposed to various treatments (normal saline, isotonic sucrose, 0.25 m sucrose, distilled water, acetate buffer pH 5.0, cold acetone, neutral formalin, acetic-ethanol, Triton X-100, hydrocortisone, choloroquine and vitamin A) was compared with that of control cells stained under identical conditions. Statistical differences in staining between the test and control cells were determined by the Wilcoxin Signed Rank Test and also by regression analysis following a transformation designed to allow for the saturation character of the reaction. The success of the modified technique depends upon meticulous methodology. It is capable of demonstrating both lysosomal membrane labilization and stabilation, second- and third-stage lysosomal activation, and apparent lysosomal enzyme loss or destruction in situ. The technique also allows the degree of reversible or first-stage lysosomal activation to be subdivided on an almost continous basis and is suitable for investigating the effects of drugs and other agents on the integrity of the lysosome in situ.


A selective stain for mast cells.

A selective stain for mast cells. A selective stain for mast cells in tissue sections is presented. The procedure is based on the resistance to destaining with absolute ethanol-acetic acid of the complex acid mucopolysaccharide-Toluidine Blue reinforced with ferrioxamine B.


Acute adverse effects of antibiotics.

Acute adverse effects of antibiotics. A review of selected clinical reports in man and experimental studies in lower animals suggests that, under certain circumstances, several commonly used antibiotics may cause cardiovascular depression, respiratory difficulties, or alter the metabolic breakdown of other drugs. These untoward responses are believed to be due to the direct effects of antibiotics on specific physiologic functions, rather than to be related to allergic reactions or cytotoxic lesions. Severe pathologic conditions, over-dosage, or concomitant exposure to other potent drugs may predispose a patient to these acute adverse effects.


Characterization of cytosine permeation in Saccharomyces cerevisiae. Cytosine permeation in Saccharomyces cerevisiae has been studied. Cytosine uptake is mediated by a permease which is also responsible for purines transport. The Km for the transport of various substrates of this permease have been determined. By means of appropriate selective techniques, mutants with altered Km and mutants lacking the permease have been selected. Cytosine transport is active and is inhibited by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, and by N-ethylmaleimide, a reagent of--SH group. Internal labeled cytosine is chased by addition of unlabeled cytosine in the medium. These results support the hypothesis of a carrier-mediated transport, with reduced internal affinity, allowing the release and accumulation of cytosine in the inner compartment. The efflux of cytosine from cytosine permease-less cells has also been studied and shows first order kinetics. A diffusion coefficient of 5.7 per 10- minus 8 cm per S- minus 1 has been evaluated for this efflux.


Tannic acid-stained microtubules with 12, 13, and 15 protofilaments. Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.


Localization and characterization of carbohydrates in adrenal medullary cells. The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.


Ultrastructural and biochemical observations on interphase nuclei isolated from chicken erythrocytes. Adult hen erythrocyte nuclei are isolated from cells or haemolysed in situ by acting on the plasma membrane with rotating knives or with non-ionic detergents. When the isolation medium contains magnesium ions (1 mM), sucrose (0-4 M) and Tris buffer (0.01 M, pH 7-5) called SMTOG (see text), the ultrastructure in thin sections through the condensed chromatin bodies, after staining with either uranyl-lead or phosphotungstic acid (PTA), is similar to that found in the intact cell. Hence it can be concluded that the 2 phases which comprise chromatin, the o- and e-phase, survive nuclear isolation. These are so called because the structural units in chromatin are arranged at the surface of the nucleus into one or more layers and give rise to oddly (o) and evenly (e) numbered bands. The 0-phase is also largely retained after extensive washing in 0-07 M NaC1 as shown by electron microscopy and biochemical measurements; only 6% of the total nuclear protein is removed, a value small compared with the fractional amount of the chromatin protein calculated to lie in the o-phase, about 70%. After extensive washing in saline-EDTA there are structural changes in chromatin, but biochemical data show that the molecules in the o-phase are also largely retained; loss of protein amounts to between 5 and 11%. These data suggest that the o-phase is a structural component of the chromatin bodies. They support the hypothesis that condensed chromatin is formed by folding superunit threads. These units consist of a central thread-like element about 17 nm diameter which stains preferentially with uranyl-lead and forms the e-phase, with an outer cylindrical shell forming the o-phase of total diameter about 28nm. The 5-10% proteins removed by salt washes are located exclusively in a particulate component, quite likely the chromatin. They have been examined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. There are about 10 or more protein species, ranging in molecular weight from 21000 upwards. The groups of large granules previously found in the nuclear sap of intact erythrocytes are shown to be associated with an amorphous or finely  fibrillar body.


Thyroid function in patients in clinical remission after medical treatment of thyrotoxicosis. This paper described the changes in the levels of serum triiodothyronine, serum thyroxine,serum thyrotrophin and other indices of thyroid function between 2-5 y after completion of antithyroid drug therapy in 35 patients who were euthyroid on clinical criteria. There was a small but significant elevation of the mean triiodothyronine and thyroxine levels with a relative hypersecretion of Triiodothyronine. No correlation was found between the levels of either thyroid hormone measured or their ratio and the radioiodine uptakes and clearance rate, the plasma inorganic iodine level, the absolute iodine uptake or the serum TSH level. There was no case of clinical hypothyroidism but in one patient the TSH level was at the upper limit of the nrmal range and an exaggerated TSH response to TRH was found.


Normal and pathological serum levels of alpha2-macroglobulins in men and mice. The serum levels of alpha2-macroglobulin have been measured in normal men and mice and in a number of immunopathological conditions. Normal human concentrations are high in youth, reach their minimum in middle age, and gradually increase with old age. In all age groups the mean is higher in the female than in the male. Conversely, in normal mice the alpha2M level is low in youth, maximum in middle age, and shows a slight depression with old age, and the levels are frequently higher in males than in females; there are also strain variations. In human immunopathological conditions, there are some deviations from the normal alpha2M level but these are seen to be changes from the normal distribution of values around the mean, rather than significant elevation or depression of mean values. In some disease states studied there are differences between the sexes in the deviation from normal. "Abnormal" strains of mice had alpha2M levels within the range exhibited by "normal" mice but changes in the levels are seen in mice with various myelomas.


Olivocochlear and vestibular efferent neurons of the feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry. Anterograde degeneration studies have shown that the cochlear and vestibular receptor organs receive an efferent innervation from neurons in the brain stem. This pathway may provide a mechanism by which the CNS could modulate its own afferent input. The neurons which provide this innervation have so far escaped positive identification with methods which depend on retrograde cell changes after axotomy. In the present study, horseradish peroxidase (HRP) was injected into the labryinths of kittens and after allowing 24 hours for the retrograde axonal transport of this tracer, its presence in neurons of the brain stem was demonstrated histochemically. Because there is evidence that the efferent innervation of the labyrinth is cholinergic, acetylcholinesterase (AChE) was also demonstrated histochemically in the same or in adjacent tissue sections. Neurons labelled with HRP were found bilaterally in most periolivary cell groups of the superior olivary complex (cochlear efferents) and in the parvocellular reticular nucleus lateral to the abducens nucleus (vestibular efferents). Counts of labelled neurons yielded estimated totals of 1,700-1,800 cochlear and 400-500 vestibular efferent neurons. Approximately 60% of the neurons in each total were located on the side ipsilateral to the injection. The distribution of HRP-labelled neurons was virtually identical to that of AChE-positive neurons found in adjacent sections, and in those regions with predominantly ipsilateral or contralateral projections, there was an approximate correspondence in number of HRP- and AChE-positive neurons. In tissue sections processed successively for demonstration of HRP and AChE, virtually all HRP-labelled neurons were found to be AChE-positive. These findings suggest that a number of current conceptions regarding labyrinthine efferent systems may need revision.


Adenylate deaminase. A method for its localization in skeletal muscle. A method for light microscopic localization of adenylate deaminase in sections of frozen rat quadriceps muscle is described. The method depends on the hydrolysis of 6-chloropurine ribonucleotide (the 6-chloroanalogue of adenylate) and the trapping of Cl minus by Ag plus. The resulting AgCl precipitate was made visible by exposing the sections to light. After this treatment black deposits about 1 mu in diameter were seen in muscle cells. These observations indicate that adenylate deaminase of rat quadriceps muscle is located at discrete sites within the muscle cells.


A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method. In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.


Histochemical localization of cytochrome oxidase in gastric mucosa. A cytochemical technique for localizing cytochrome oxidase activity, based upon the oxidative polymerization of 3,3-diaminobenzidine (DAB) to an osmiophilic reaction product, has been employed to test the possibility of extramitochondrial cytochrome oxidase in gastric mucosa. Deposition of reaction product was found to be exclusively localized within mitochondris; in particular, no reaction product was observed at the apical plasma membrane. Measurements of the effect of DAB on acid secretion revealed a biphasic action consisting of an initial stimulation followed by inhibition. The stimulation of secretion of DAB is interpreted to indicate that DAB is oxidized via a pathway which is linked to the secretory process. The combined cytochemical and physiological measurements provide evidence that the metabolic energy supply for acid secreation is derived from mitochondrial reactions. The results are discussed in relation to current models for the coupling between acid secreation and oxidative metabolism.


Differential feulgen-deoxyribonucleic acid hydrolysis patterns of Herpes simplex virus type 1 and type 2 infected cells. Infection of human embryonic lung cells with herpes simplex virus type 1 (HSV-1) and herpes simplex type 1 (HSV-2) resulted in: (a) qualitative (nuclear cytopathologic) alterations and quantitative (nuclear area) differences in infected compared to control nuclei; (b) increased Feulgen-deoxyribonucleic acid (F-DNA) amounts in infected cells, probably due to viral DNA; (c) higher F-DNA levels in HSV-2 infected cells; and (d) increased rates of F-DNA hydrolysis in viral-infected as compared to uninfected nuclei.


Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation. Endogenous mannary gland peroxidase in acinar cells of prelactating and lactating rats is revealed in tannic acid-formaldehyde-glutaraldehyde-fixed tissue by means of the standard diaminobenzidine procedure. Diaminobenzidine cytochemical reaction product is present in perinuclear cisternae, in the granular endoplasmic reticulum and in Golgi apparatus of functionally differentiated secretory cells. The mammary gland peroxidase is thought to represent lactoperoxidase. Peroxidase staining is diminished or absent in acinar cells of hypophysectomized and ovariectomized rats, in normal rats during early pregnancy and in nonpregnant mature females. Endogenous peroxidase or a heme protein with peroxidatic activity may be considered an ultracytochemical marker enzyme for acinar cells actively engaged in lactogenesis.


The role of T cells in IgG production; thymus-dependent antigens induce B cell memory in the absence of T cells. B cell memory was shown to develop in congenitally athymic (nu/nu) mice after injection with small amounts of thymus-dependent antigens, in particular heterologous serum proteins, such as fown gamma-globulin (FGG) or DNP-bovine-serum albumin (DNP-BSA). Large doses of proteins (10 mg) tended to produce a specific B cell unresponsiveness, although there was still some evidence of B cell priming. The antigen did not have to be in a multivalent form to interact with B cell so as to induce immunologic memory or tolerance. In contrast to the induction of B cell memory, the production of IgG antibody in this system was found to be strongly T cell dependent. Thymus-independent antigens like LPS or POL with pronounced adjuvant effects on IgG production in normal or surgically thymectomized mice, could not replace T cells in allowing an IgG response against thymus-dependent antigens in congenitally athymic mice. However, the action of T cells once activated is likely to be non-antigen-specific, since it was shown that supernatants of antigen-activated-syngeneic T cells stimulated IgG production in cultures of primed B cell populations non-antigen-specifically.


The mechanism of basophil histamine release induced by antigen and by the calcium ionophore A23187. The mechasism of human basophil histamine release by the calcium ionophore A23187 has been compared to that induced by the interaction of antigen with cell bound IgE antibody. Ionophore induced histamine release (Ion. H.R.) occurs with the leukocytes of both normal and allergic donors. It is completely calcium dependent; LaCl3 inhibits both Ion. H.R. and antigen induced histamine release (Ag. H.R.) at about 10-minus 7 M. The kinetics of Ion. H.R. suggest that this process has no "desensitization" phase as does Ag. H.R. and the ionophore is fully active on antigen-desensitized cells. Pharmacologic studies indicate that dibutyryl cyclic AMP and agents which increase endogenous cyclic AMP levels do not inhibit Ion. H.R. as they inhibit the early stages of Ag. H.R. Of the agents which affect microtubules, colchicine inhibits and D2O enhances Ion. H.R. in a manner which is qualitatively similar but quantitatively less marked than their effects on Ag. H.R. The metabolic antagonist 2-deoxyglucose inhibits both Ion. H.R. and Ag. H.R. in a similar fashion. Based on these data and the observation that cells pretreated with ionophore show a marked (synergistic) enhancement of Ag. H.R. we conclude that Ion. H.R. has a similar or identical mechanism to the later stages if Ag. H.R. but "short circuits" the cyclic AMP-associated events of Ag. H.R.


Conformation-dependent antigenic determinants in the toxic lectin ricin. The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.


Effect of anti-beta2-microglobulin on antigen and allogeneic lymphocyte-induced proliferation of human lymphocytes. The effect of anti-beta2-microglobulin (beta2m) on the mixed lymphocyte reaction (MLR), and an antigen-induced proliferative response was studied. Anti-beta2m IgG and Fab' fragments completely inhibited the MLR. Preincubation of stimulator or responder cells with anti-beta2m suggested that the major effect of anti-beta2m may be on the responder cell population. A clear-cut effect on responder cells was demonstrated by showing that anti-beta2m completely inhibited a MLR in which the stimulator population was a beta2m negative lymphoblastoid cell line. Anti-beta2m also inhibited PPD-induced proliferation of sensitized lymphocytes. The kinetics of this inhibition indicated that anti-beta2m added within the first 18 hr of stimulation was effective in inhibiting the proliferative response. These data are discussed in light of the hypothesis that beta2m may be a subunit of an antigen receptor on T cells.


Preventive effect of hapten-reactive thymus-derived helper lymphocytes on the tolerance induction in hapten-specific precursors of antibody-forming cells. The role(s) of helper T lymphocytes in preventing or altering tolerance induction in DNP-specific B lymphocytes was studied. As DNP-reactive helper T cells were reactive against the DNP-portion of the DNP-D-GL molecule, we could probe definitively the physiological role of helper T cells in preventing tolerance induction in B lymphocytes by DNP-D-GL. The results demonstrated that the induction of DNP-specific B cell tolerance by DNP-D-GL can be completely prevented by the presence of DNP-reactive helper T cells, and provide evidence that one critical role of helper T cell participation in humoral responses to antigens is to circumvent the development of a tolerogenic signal that, in the absence of such T cell function, might otherwise ensue after binding of the antigenic determinants by specific B lymphocytes.


Hepatitis B core antigen. Detection of antibody by radioimmunoprecipitation. Radioactive cores were prepared from concentrated Dane particles by DNA polymerase reaction followed by CsCl density gradient centrifugation. Two radioactive peaks were obtained: one peak with an average density of 1.36 g/cm-3 in CsCl contained cores that possessed full serologic reactivity; the other peak, with a density of 1.28 to 1.32 g/cm-3, contained cores associated with globulin. A double antibody immunoprecipitation test was developed, using the radioactive heavy cores as a source of antigen. The test was at least 300 times as sensitive as complement fixation for detecting antibody to core.


Biologic significance of disulfide bonds in human IgE molecules. E myeloma protein, PS, was reduced in different concentrations of dithiothreitol (DTT) for 1 hr followed by alkylation with 14C-iodoacetamide. The affinity of the reduced-alkylated molecules for target cells was evaluated by their ability 1) to sensitize primate skin in a reversed P-K reaction, 2) to sensitize human basophils in a reversed-type histamine release and 3) to block passive sensitization with reaginic antibody. Antibody-epsilon0 antibody was employed for reversed type reactions to avoid participation of cell-bound normal IgE in the reactions. The sensitizing activity of IgE did not change following reduction in 1 mM DTT, which split inter-heavy-light chain disulfide bond. The activity of IgE significantly diminished after reduction in 2 mM DTT followed by alkylation. This treatment resulted in the cleavage of two intra-epsilon-chain disulfide bonds, which are present between the hinge and the Fd portion of the molecules. The reduced-alkylated protein was capable of sensitizing primate skin and human basophils, however, a much higher concentration of the reduced-alkylated protein than the native protein was required for passive sensitization. The optimal sensitization period for the reversed P-K reaction was 3 hr with the reduced-alkylated protein. The protein had the ability to block passive sensitization with reaginic antibody. The reduced-alkylated protein and the native protein were labeled with 125I, and binding of these proteins with human basophils was examined by autoradiography. The results showed that affinity of the reduced-alkylated protein for basophils was less than that of native protein. Since the disulfide bonds split by 2 mM DTT were not included in the Fc portion of the molecules, the Fc fragment was obtained from the reduced-alkylated protein and was tested for affinity for basophils. It was found that the Fc fragment had higher affinity than the reduced-alkylated protein. Recovery of the affinity by papain digestion strongly suggested that cleavage of disulfide bonds in the Fab portion of the molecules induced conformational changes in the Fc portion which is involved in binding to the target cells. Reduction of IgE with 10 mM DTT followed by alkylation resulted in cleavage of 5 disulfide bonds, which is accompanied by a loss of both sensitizing and blocking activities. The fifth disulfide bond which was cleaved by 10 mM DTT, but not by 2 mM DTT, appears to be an inter-heavy chain disulfide bond in the Fc portion of the epsilon-chains. Neither epsilon1 nor epsilon2 determinants in the Fc portion of epsilon-chains were degraded by this treatment.


Early and enhanced antioxin responses elicited with complexes of tetanus toxoid and specific mouse and human antibodies. Primary tetanus antitoxin responses were early and enhanced in mice when tetanus toxoid was administered in complex with specific isologous antitoxin or specific mouse gamma-globulin. Antoxin responses were enhanced when fluid tetanus toxoid was complexed in vitro in antigen-to-antibody ratios of equivalence or antigen excess; responses to complexed toxoid in antibody excess were comparatively repressed. Primarly responses were greatly inhibited in mice immunized with the same amount ot toxoid complexed in vitro in antigen-to-antibody ratios of equivalence or antigen excess; responses to complexed toxoid in antibody excess were comparatively repressed. Primary responses were greatly inhibited in mice immunized with the same amount of toxoid complexed at equivalence or in antibody excell with specific human gamma-globulin. Although primary responses. Separate injections of antigen and antibody at different sites produced an excelldnt antitoxin responses. Separate injections of antigen and antibody at different sites produced an excellent in vivo primed state for early and high responses. Antibody production after stimulation with complexed toxoid was also enhanced in mice irradiated with 400 rad, a dose that ordinarily completely suppresses primary responses with fluid toxoid alone. These data provide evidence for the efficacy of antigen-antibody complexes in early and active immunization.


South-to-north gradient in distribution of the r determinant of hepatitis B surface antigen in Japan. A total of 2.305 sera positive for hepatitis B surface antigen were obtained from asymptomatic carriers throughout Japan and subtyped for the d, y, w, and r specificities by the hemagglutination inhibition method. Determinant d prevailed in Japan (d, 99.1%; y, 0.9%), and there was no regional variation in its occurrence. In sharp contrast, there was a marked variation in the distribution of the w and r determinants, forming an apparent south-to-north gradient of r. The percentage of r determinant was highest in Kyushu (92%-94%); it decreased gradually along the axis of Japan to the north and was lowest in Akita (46%), which is located at the north end of Honshu.


The basis, technique and indication for cryosurgery in tumours of the oral cavity and face. The fundamentals of clinical application of crysurgery with very deep freezing of tissues is based on the necrotising effect of extreme cooling. The freezing of extracellular and intracellular free fluid leads to irreversible cell damage, because of a shift of electrolyte concentration to toxic values, and a direct lesion of internal cellular structures. The lesion due to cold occurs as a clear demarcation from the healthy surrounding, almost completely painlessness, and rapid healing without complication. For clinical application in the head and neck, there are cooling instruments fitted with specially shaped, exchangeable probe heads. Convincing results were achieved in the cryosurgical treatment of leucoplakias and Bowen's disease of the oral mucosa. The curative possibilities in carcinoma of the oral mucosa is small because of the limited performance of the cryo-carrier, which is best suited for superficial, non-infiltrating tumours. Preoperative rapid cooling of melanomas was performed to avoid dissemination of tumour cells during resection. Favourable functional results were achieved in the treatment of hemangiomas.


The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex. Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.


Inheritance of antibody specificity. II. Anti-(4-hydroxy-5-bromo-3-nitrophenyl) acetyl in the mouse. Mice of 17 inbred strains produced anti-(4-hydroxy-5-bromo-3-nitrophenyl)-acetyl (NBrP) of three different fine specificity types. Anti-NBrP antibodies of all allotype b mice (five strains tested) had a high relative affinity for (4-hydroxy-3.5-dinitrophenyl) acetyl (NNP) but low for (4-hydroxy-5-cloro-3-nitrophenyl) acetyl (NCP). Another category was characterized by high relative affinity for NCP but low for NNP. This category included most of the tested strains. The third category (CBA and C3H strains) had an intermediate fine specificity. Associated with fine specificity characteristics were anti-NBrP titers, mice of allotype b had lower titers than the other mice. Studies of congenic, recombinant inbred, F1 and backcross mice showed that both fine specificity and the magnitude of the anti-NBrP response of tbalb/C MICE WERE CONTROLLED BY AN ALLOTYPE-LINKED GENE. This gene was dominant over the C57BL/6 ALLELE. Lack of recombinant mice in the backcross generatioterns on the other suggest close linkage between the two genes.


Functional heterogeneity of lymphocytic choriomeningitis virus-specfic T lymphocytes. I. Identification of effector amd memory subsets. At varying intervals after immunizing infections of adult BALB/c mice with lymphocytic choriomeningitis (LCM) virus, splenic lymphocytes were tested for their ability to either elicit acute LCM or protect against lethal intracerebral LCM virus challenge when transferred to syngeneic recipients that were, respectively, virus carriers induced by cyclophosphamide-induced immunosuppression and normal susceptible mice. These lymphocytes were also assayed for their capacity to lyse, in vitro, LCM virus-infected BALB 3T3 cells labeled with 51Cr. Only lymphocytes obtained from donor mice between 6 and 8 days postimmunization significantly protected normal recipients from the development of lethal central nervous system (CNS) disease when transferred 18-24 h before virus challenge. At 10 days they were not protective even though their cytolytic activity in vitro either exceeded or approximated that of protective lymphocytes. The capacity to protect more closely corresponded with a period of virus-induced DNA synthesis in donor spleens as measured by the incorporation of [5--125I]-2'-DEOXYURIDINe. However, none of these cytolytically active lymphocyte populations were effective in mediating acute CNS disease when transferred to virus-carrier mice. In contrast, lymphocytes obtained 18 days or later after primary immunization, although having no protective capacity and exhibiting minimal cytolytic activity in vitro, were able to regularly produce acute disease in virus-carrier mice. The ablation by anti-theta-serum treatment of these in vivo and in vitro virus-specific immune functions established that the relevant lymphoid cells were T lymphocytes. Similarly, reconstitution of C3H times C57BL F1 adult mice, depleted of lymphocytes by thymectomy and lethal irradiation, with syngeneic day 8 immune donor lymphocytes rendered them resistant to the development of the carrier state after intracerebral virus challenge; reconstitution of such animals with nonimmune lymphocytes restored their ability to develop typical LCM. Collectively, these data indicate that immunogenic stimulation with LCM virus leads to the development of at least two different virus-specific T-lymphocyte subsets: (a) an early appearing transient effector population of cells and, perhaps, their immediate precursors whose generation requires the presence of virus, and (b) a stable memory population that appears well after virus clearance and which has little or not no cytolytic activity. The possible mechanism by which these functionally different subsets can influence the outcome of acute or chronic LCM virus infections is discussed.


Isolation and analysis of the mechanism of action of an inactivator of C4b in normal human serum. A complement regulatory principle, C4b inactivator, was isolated in a partially purified form from normal human serum. The C4b inactivator, a beta1-globulin with an approximate mol wt of 88,000 daltons, and which may be identical to C3b inactivator, cleaved C4b in free solution or on the surface of cells and rendered it unable to participate in hemolytic reactions or to interact with cells, having receptors for C4b. C/b inactivator functioned by cleaving the alpha-polypeptide chain of C4b at a single site which was sufficient to dissociate the molecule into two fragments, C4c and C4d, and to inactivate it biological function. Certain structural correlates of C4 functions deriving from these studies are discussed and a model for C4 structure based on these findings is presented.


Evidence for the clonal abortion theory of B-lymphocyte tolerance. This paper deals with the behavior of adult mouse bone marrow cells placed in tissue culture with or without antigen, and subsequently assessed for immune competence after adoptive transfer into lethally X-irradiated, syngeneic hosts. Attention was focussed on B lymphocytes through using hapten human gamma globulin (HGG) preparations as putative tolerogens in tissue culture, the T-cell-independent antigens DNP-POL and NIP-POL as challenge injections in adoptive hosts, and numbers of hapten-specific PFC in host spleens for the quantitation of immune competence. It was found that the capacity of bone marrow cells to mount an adoptive immune response rose by a factor of about fivefold over 3 days in tissue culture. This rise was completely abolished by the presence in the culture of hapten-HGG conjugates with about one mole of hapten per carrier molecule. The prevention of the emergence of immune competence amongst maturing B cells was termed clonal abortion tolerogenesis. Dose-response studies showed the lowest effective antigen concentration to be between 2.5 times 10- minus 10 and 2.5 times 10- minus 9 M, and a standard concentration of 2.5 times 10- minus 8 M was chosen as producing near maximal effects. The tolerance was antigen-specific and time-dependent, being maximal only when antigen was present continuously as the cultured cells was maturing. It did not depend on the presence of T lymphocytes in marrow, and was not of an "infectious" type. In contrast to tolerogenesis of mature B lymphocytes by high antigen concentrations, it could not be abolished by lipopolysaccharide. We speculate that clonal abortion may be a tolerance mechanism of great physiological significance for self-recognition, and discuss the results in the framework of other recent tolerance models, including those involving receptor blockade and suppressor T cells.


Recognition by pregnancy serums of non-HL-A alloantigens selectively expressed on B lymphocytes. A group of alloantibodies are found in pregnancy sera which react with antigens present on B lymphocytes and monocytes but are not detectable on the vast majority of unstimulated T cells. This specificity distinguishes them from HL-A antibodies which react with both cell types. They were readily recognized through indirect fluorescent antibody analysis by employing the combination of B-cell lymphoid lines and normal peripheral blood T cells. Different sera gave a variety of patterns of reactivity with a panel of 11 lymphoid lines. Similar differential patterns were also observed with normal B cells from different individuals particularly after concentrating the B cells. The antibodies were also cytotoxic to B cells and this procedure gave parallel results to the fluorescence method. The pattern of reactions obtained indicated a very heterogeneous system similar to that for HL-A. Special study of certain of the sera provided evidence that the lymphocyte-defined determinants of the mixed lymphocyte reaction system were involved. For convenience the term HL-B has been employed for these antigens.