Serum concentration of human alpha 2 HS glycoprotein during the inflammatory process: evidence that alpha 2 HS glycoprotein is a negative acute-phase reactant. A nonspecific opsonin function has been ascribed to human alpha 2 HS glycoprotein. Its serum level has been shown to be decreased in trauma patients. Recent studies from this laboratory revealed a heterogeneity among the products obtained in the course of the preparation of the protein. To date, no definitive agreement existed with regard to a molecular homogeneous entity of alpha 2 HS glycoprotein (Ba-alpha 2 glycoproteins). The purpose of the current work was to study the variations in serum level of alpha 2 HS in patients suffering from an acute inflammatory process of bacterial etiology and to determine whether a decrease in alpha 2 HS was accompanied by the appearance of fragments of this protein in the serum. A method of preparing alpha 2 HS was thus developed, using an immune absorbent as a final purification step. In an intermediary step of the preparation, alpha 2 HS was found to bind zinc when metal chelate affinity chromatography was employed. Immunologically and physico-chemically pure alpha 2 HS was obtained. The protein consists of a unique polypeptide chain of about 50,000 daltons and has a unique amino-terminal residue, alanine. However, the protein maintained its molecular integrity with difficulty, and spontaneous fragments ranging from 30,000 to less than 10,000 daltons were produced in some of the preparations. No major modification in the molecular structure of the protein was noted in the sera of subjects suffering from an acute inflammatory process. Serum level of alpha 2 HS and alpha 1 antitrypsin (AT)was determined in 23 patients. When the acute-phase (AP-)reactant alpha 1 AT was increased (difference with normal mean greater than +2 or +3 SD), the sera showed a large decrease in alpha 2 HS (difference with normal mean less than -2 or -3 SD). The serum level of alpha 2 HS, albumin, alpha 2 macroglobulin, and of positive AP-reactants, orosomucoidinal study of seven patients. The results were submitted to a principal components analysis. Alpha 2 HS showed a negative correlation with the AP-reactants alpha 1 AT, orosomucoid, and haptoglobin (P less than 0.05) and a positive correlation with albumin (P less than 0.05); these findings indicate that alpha 2 HS is a negative AP-reactant. In addition, analysis of the principal components confirms thestrong analogy between alpha 2 HS and albumin and indicates that serum level behavior of the AP-reactants during the course of the disease closely depends on the protein studied.


Combined functional and immunochemical analysis of normal and abnormal human factor X. Human Factor X was isolated from Cohn fraction III and characterized by polyacrylamide gel electrophoresis, amino acid composition, and isoelectric focusing. Two molecular forms with biological activity were observed at isoelectric points of 4.8 and 5.0. Antisera generated to Factor X was monospecific and used to establish an equilibrium competitive inhibition radioimmunoassay. This assay was specific for human Factor X and did not cross-react with human prothrombin or bovine Factor X within the sensitivity range of 6-300 ng Factor X antigen/ml. The mean concentration of Factor X based on the antigen was 11.9 mug/ml, whereas concentration values based on coagulant activity was 7.8 mug/ml. This 30% difference in measurement appears to result from the presence of a subpopulation of Factor X molecules devoid of coagulant activity. The radioimmunoassay was used to qualitatively and quantitatively compare purified Factor X to plasmic Factor X obtained from normal, warfarintreated, acquired Factor X-deficient, and congenitaldeficient patients. In all but one case, the Factor X present in these plasmas was immunochemically identical to the purified Factor X and permitted precise quantitation of these abnormal Factor X molecules. Factor X procoagulant activity was analyzed relative to Factor X antigen and the specific activities were used to characterize normal and abnormal Factor X molecules. Reduced Factor X activity in plasmas from warfarin-treated and acquired Factor X-deficient patients was attributed to both decreases in Factor X antigen and decreased function of the Factor X molecules. Congenitally deficient patients, in general, showed a reduction in Factor X antigen in parallel with Factor X procoagulant activities resulting from comparable decreases in specific biological activity of the molecules.


Scleroderma: increased biosynthesis of triple-helical type I and type III procollagens associated with unaltered expression of collagenase by skin fibroblasts in culture. To assess potential abnormalities in collagen metabolism in systemic scleroderma, skin fibroblast lines from patients with this disease were established and compared to control cell lines derived from healthy subjects. For studies on the biosynthesis of procollagen, the cells were incubated with [(14)C]proline in a medium supplemented with ascorbic acid and beta-aminopropionitrile, and the synthesis of nondialyzable [(14)C]hydroxyproline, in relation to DNA or cell protein, was taken as an index of procollagen formation. Five of eight scleroderma fibroblast cell lines demonstrated procollagen biosynthesis rates significantly higher than the controls, and the mean rate of procollagen synthesis by scleroderma fibroblasts was about twice that of the control cells. Control experiments demonstrated that the specific activity of the intracellular free proline was not different in scleroderma and control fibroblasts, and the mean population doubling times of the scleroderma and the control fibroblast cell lines were the same. The relative synthesis of the genetically distinct procollagens was examined by isolating type I and type III procollagens from the cell culture medium using DEAE-cellulose chromatography. The ratios of type I/III procollagens in scleroderma cell lines did not differ from the controls. The helical stability of the collagenous portion of type I and type III procollagens, estimated by the resistance of (14)C-collagen to limited proteolytic digestion with pepsin under nondenaturing conditions, was the same in both scleroderma and control cultures. The capacity of the cells to synthesize enzymatically active and immunologically reacting collagenase was also studied; no marked differences in these parameters could be observed. The results suggest that cultured skin fibroblasts from patients with scleroderma demonstrate a metabolic abnormality expressed as increased synthesis of type I and type III procollagens in a normal ratio. This abnormality may play a role in the excessive accumulation of collagen in the skin and other organs affected in scleroderma.


Public antigenic determinant on a family of HLA-B molecules. Serologic cross-reactivity among allelic gene products commonly occurs in the HLA complex, but the molecular basis of these serologic phenomena is incompletely characterized. Because of strong cross-reactivity among antigens comprising the B7 cross-reactive group (i.e., HLA-B7, Bw22, B27, B40, and Bw42) and because of the association of several antigens of this group with spondyloarthropathies, we initiated a study of the chemical basis of cross-reactivity among this group of antigens. Using classic serologic procedures, 125I-Protein A binding assay, and chemical immunoprecipitation techniques, we have defined a new antigenic determinant, tentatively designated "X", which is present on certain HLA-B molecules. By a series of sequential immunoprecipitation experiments, X was shown to be a "public" antigenic determinant distinct from the "private" determinants B7, Bw22, B27, and B40, but present on the same 44,000-dalton glycoprotein molecules. The implications of this finding regarding disease predisposition and HLA typing as a diagnostic aid are discussed.


Stimulation of neutrophil oxygen-dependent metabolism by human leukocytic pyrogen. The ability of highly purified human leukocytic pyrogen (LP) to stimulate neutrophil oxygen-dependent metabolism was studied. Human peripheral blood neutrophils exposed to leukocytic pyrogen in vitro demonstrated an increase in the percentage of neutrophils reducing nitroblue tetrazolium (NBT) dye and a marked stimulation of superoxide dismutase inhibitable reduction of ferricytochrome c. LP stimulation of neutrophil oxygen-dependent metabolism was dose and time dependent. Procedures that destroyed the pyrogenicity of LP also abolished the effects on neutrophil metabolism. Neutrophil hexose monophosphate shunt activity was also stimulated by LP. In a rabbit model, the effect of in vivo LP on neutrophil superoxide generation was also studied. There was a consistent increase in the percent and absolute number of NBT positive neutrophils. Peak stimulation of neutrophil metabolism occurred after defervescence suggesting several possible mechanisms. The observations reported here may, in part, explain the nonspecificity of the NBT test in febrile, noninfected patients and provide further understanding of neutrophil physiology during acute inflammation.


Inhibition by non-steroidal anti-inflammatory agents of the ascorbate-induced elevations of platelet cyclic AMP levels. Ascorbate induces a 10- to 25-fold rise in platelet guanosine 3'5'-cyclic monophosphate (cGMP) and this action is prevented or reversed by the introduction of aspirin, indomethacin, ro 5,8,11,14-eicosatetraenoic acid (TYA). The reversal was 70-90% complete at 30 s, the earliest time point that was examined. As the effect of ascorbate on cGMP was not diminished by anaerobic conditions, which inhibited the oxidation of exogenous arachidonate by more than 95%, metabolic inhibition of cyclo-oxygenase activity did not duplicate the effect of the non-steroidal anti-inflammatory agents. Ascorbate did not act by the activation of phospholipase A2 in that the ascorbate-induced evevation of cGMP was not accompanied by increased oxygen consumption or the release of [14C]-arachidonate from prelabeled platelets. Thus, despite the finding that non-steroidal anti-inflammatory agents prevent and reverse the ascorbate-mediated elevation of cGMP, it was not possible to relate their respective antagonist and agonist actions to the oxidation of arachidonate.


Histamine release and complement changes following injection of contrast media in humans. Mechanisms responsible for allergic-like reactions following administration of radiographic contrast media (RCM) are unclear. Aortic root blood specimens were obtained sequentially in 6 subjects following injection of RCM into the pulmonary artery during cardiac catheterization. In 5 subjects, elevated plasma histamine levels (up to 80 ng/ml) occurred within minutes. Levels of C3, C4, factor B, and total hemolytic complement activity were decreased in the same specimens. No hemodynamic or clinical abnormalities were noted. These findings support the concept that RCM can liberate histamine in vivo in humans. Complement alterations may be related to localized RCM-protein interaction. It is unclear whether complement changes are related to the RCM-induced allergic mediator release.


[The level of alpha-feto protein in amniotic liquor. The antenatal diagnosis of some malformations (author's transl)]. We have measured the levels of alpha-feto protein in 127 samples of amniotic fluid, obtained in the course of antenata diagnosis, using the immunoelectrophoretic method of Laurell. The mean, the upper limit of normal and the extreme levels of alpha-feto protein in liquor have been calculated for each gestational age. The difficulties which have been met with in interpreting the results (false positives and false negatives) have been discussed.


Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport. We have compared the retrograde axonal transport of horseradish peroxidase (HRP), to the retrograde transport of HRP conjugated with wheat germ agglutinin (WGA). Morphometric studies have shown that WGA-HRP conjugates were 40 times more sensitive than free HRP, in the tracing of retrograde connections from the rat submandibular gland to the superior cervical ganglion. Also, WGA-HRP was more sensitive than free HRP in the tracing of retrograde connections from the rat tongue to the hypoglossal nucleus. Our findings with WGA-HRP are consistent with the observations by Schwab et al. who reported (-125I) WGA is a highly sensitive retrograde tracer (Brain Research 152:145, 1978 (22)).


Staining properties of aldehyde fuchsin analogs. This investigation was designed to clarify the role of the aldehyde component of aldehyde fuchsin in its staining reactions. Several aldehyde fuchsin analogs were prepared by using different aldehydes. The staining quality of these analogs and pararosaniline-HCl was compared with that of aldehyde fuchsin prepared with paraldehyde in the usual way. The major findings of this investigation include: 1) Aldehyde fuchsin staining of nonoxidized pancreatic B cells requires a stain prepared with either paraldehyde or acetaldehyde. 2) An aldehyde moiety is required for aldehyde fuchsin staining of strong tissue anions. 3) Staining of elastic tissue with aldehyde fuchsin analogs resembles staining of strong tissue anions more than staining of nonoxidized pancreatic B cells. Possible reaction mechanisms of aldehyde fuchsin with tissue substrates are discussed.


The cationic carbocyanine dyes Stains-all DBTC, and Ethyl-Stains-all, DBTC-3,3',9 triethyl. A simple method is presented for distinguishing two closely related metachromatic carbocyanine dyes: Ethyl-Stains-all, a triethyl dye, and Stains-all, a diethyl methyl dye. This has become important since one lot of the triethyl dye was distributed erroneously under the diethyl methyl label. The dyes differ in solubility and in differential staining of macromolecules. Studies performed with both dyes are summarized.


Barrier filter for fluorescence microscopy of strongly autofluorescent plant tissues. Application to actin cables in Chara. A liquid barrier filter for use in fluorescence microscopy of strongly autofluorescent plant tissues is described. The filter consists of a methanol solution of cupric chloride and ferric chloride and isolates fluorescein fluorescence from the strong red autofluorescence of photosynthetic plant tissues. Subcortical actin cables in the giant alga Chara are being visualized through use of this filter together with heavy meromyosin labeling.


Cellular origins of different forms of gastrin. The specific immunocytochemical localization of related peptides. We have localized the antigenic determinants for the main forms of gastrin (big gastrin, G34, and little gastrin, G17) in hog antral mucosa using sequence specific antibodies and an indirect immunofluorescence technique. Populations of monospecific antibodies were obtained after affinity immunoadsorption to remove populations of unwanted specificity. The specificity of the purified antisera was established by direct binding of 125I labeled peptides to antisera at the same dilutions as those used in immunocytochemistry. The results indicate that in hog antral mucosa there is a single population of cells with the antigenic determinants of the C-terminal region of G17 and G34, the N-terminal region of G17, the N-terminal region of G34, and the intact G17 molecule. In duodenum there are cells with only C-terminal reactivity; since gastrin and CCK share a common C-terminal sequence it is concluded that this cell type contains CCK-like peptides rather than gastrin.


The histochemical distribution of protein bound sulfhydryl groups in human epidermis by the new staining method. Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) which is nonfluorescent by itself but will react readily with -SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of -SH groups and S--S linkages in the human epidermis. The distribution of -SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S--S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared gradually. The distribution of S--S linkages appeared to be very low in the cytoplasm of keratinized cells. No substantial fluorescence was localized on keratohyalin granules even after reduction.


Development of a new primary fixative for electron microscopic immunocytochemical localization of intracellular antigens in cultured cells. We have developed a new primary fixative that permits the localization of intracellular antigens with well preserved ultrastructural morphology. This primary fixation method employs a mixture of a water soluble carbodiimide with glutaraldehyde, and preserves morphology, yet produces a permeable cytosol matrix so that antibodies can gain access to fixed proteins. Cultured cells were primarily fixed, treated with detergent to permeabilize their membranes, reacted with peroxidase labeled antibodies, secondarily fixed, and embedded in situ. The variations in morphology and accessibility of intracellular antigens were evaluated for a variety of fixatives. Concanavalin A and alpha 2 macroglobulin were chosen as examples of intracellular protein antigens to evaluate these fixation methods. Both of the proteins were localized in intracellular vesicles.


Immunoferritin location of carbamoyl phosphate synthetase in rat liver. Experiments were carried out to locate carbamoyl phosphate synthetase (CPS) in rat liver by direct immunoferritin labeling. By using Epon sections treated with sodium methoxide, homogenates or mitochondrial and mitoplast fractions, carbamoyl phosphate synthetase was found homogeneously distributed in the mitochondrial matrix. Immunoferritin was detected with high resolution which permits the identification of individual molecules. Measurements were made of the number of ferritin particles per square micron of mitochondrial surface, providing a novel and independent assessment of the carbamoyl phosphate synthetase concentration.


Enzymatic tracers in the study of vascular permeability. Elucidation of the ultrastructural basis of vascular permeability was aided by the development of cytochemical techniques for visualizing the distribution, within the vessel wall, of intravenously injected peroxidatic enzymes of varying molecular size. Tracer enzymes available range from 10 A (hemeoctapeptide) to 52 A (catalase) effective molecular radius. The use of enzymatic probe molecules assumes a thorough characterization of: (a) the molecular charge (isoelectric point of the native enzyme, and when feasible, its polyanionic and polycationic derivatives; (b) effective molecular radius (ae); (c) peroxidase activity (to detect by spectrophotometry of DAB-oxidizing activity, the optimal pH, temperature, and enzyme concentration to be employed in the cytochemical procedure). Molecular shape and state of dispersion of the enzymatic probes should be determined by gel chromatography and spectrophotometry of both the tracer solution and aliquots of blood plasma collected after i.v. injection of the tracer. Conditions required for the probe administration include: (a) the investigation of potential side effects (tests for toxicity and vascular leakage) and (b) estimation of the tracer volume and concentration which does not affect significantly the blood volume and osmotic pressure. Determination in vitro of the crosslinking of tracer molecules induced by the aldehyde fixative to be employed, also gives an indication on potential diffusion artifacts. Based on the information thus obtained, the design of the cytochemical procedure should also take into account the possible use of methods for enhancing the peroxidatic reaction product: nitrogenous ligands (imidazole, diaminopyrimidine, histidine) or polyphenolic mordants (galloylglucoses). The usefulness of peroxidatic tracers in the investigation of vascular permeability is exemplified by some results obtained on the microvascular endothelium in vivo (trasncytosis, intercellular pathway, etc.), and on endothelial cells isolated from heart microvasculature.


The use of avidin-biotin interaction in immunoenzymatic techniques. Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.


Clinical applications of the enzyme labeled antibody method. Immunoperoxidase methods in diagnostic histopathology. Histological criteria for the definition of disease entities have largely been established with light microscopy of conventionally stained and routinely processed tissue sections. More or less specific histochemical staining procedures and more recently enzyme-histochemical and quantitative histo- and cytochemical techniques in some cases provided additional criteria. In the last decade, however, the introduction of immunofluorescence and more recently the different immunoperoxidase methods have significantly influenced the scope of contemporary histopathology. Especially, the possibility to use immunoperoxidase methods on routinely processed tissue specimens has offered new dimensions in diagnostic pathology. These methods proved of particular importance for: 1) The development of new criteria for diagnosis, prognosis and treatment (e.g. immunological classification of lymphoma; plasmacell typing in intestinal inflammatory conditions; human chorionic gonadotropin and alpha-fetoprotein in germ cell tumors of the testis). 2) The possibility of etiological diagnosis (e.g. the recognition of hepatitis B viral antigens in liver biopsy specimens; histological typing of causative micro-organisms in inflammatory conditions). 3) The recognition of disease entities that were hitherto unrecognized (e.g. hyperplasia of parafolicular C-cells in the thyroid in multiple endocrine neoplasia syndromes; gastric G-cell hyperplasia as a variant of Zollinger-Ellisons syndrome). 4) Functional analysis of tissue components (e.g. hormone content of pituitary and pancreatic adenomas; cytoplasmic differentiation produces in "undifferentiated" tumors). It can be expected that immunoenzymehistochemistry will soon play a major role in routine diagnostic histopathology.


The role of sialated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium. Changes in the anionic charge distribution at the luminal face of the endothelium of the sinusoids of the bone marrow have been studied at sites of endocytosis by large bristle coated vesicles and at the sites of molecular permeability through diaphragmed fenestrae. The anionic charge distribution has also been studied at the abluminal aspect of these vessels at sites of transmural blood cell passage. Cationic surface markers such as colloidal iron, native ferritin and polycationic ferritin used at low pH, 1.8, and the use of neuraminidase show that the nonmodified endothelial cell surface has exposed sialic acid groups, which are absent at the sites of these functional specializations. Polycationic ferritin binding over a range of pH levels indicates the prsence of another species of anionic materials present at both the nonmodified cell surface and at the sites of the cell surface modifications. This second group of anionic compounds is neuraminidase resistant and has a pKa higher than that of sialic acid (pKa:2.6).


The histochemistry of colonic mucins.

The histochemistry of colonic mucins. This presentation could perhaps better be titled the histochemistry of colonic mucins, as based upon PAS reactivity, since it mainly concerns development of methods for the identification of the various patterns of o-acylation of sialic acid as measured by alteration in PAS staining.


Anomolous staining patterns in immunohistologic studies of malignant lymphoma. A number of immunoperoxidase studies of malignant lymphomas have reported polytypic light chain staining of neoplastic cells, thus bringing into question the concept that the monoclonality of B cell lymphomas is reflected in their synthesis of monotypic light chain. In this study of a large number of Ig positive lymphomas, staining for a wide variety of antigens has identified clear differences between monotypic Ig synthesizing cells and cells staining polytypically which appear to be taking up Ig from the environment. Attention to the nature of Ig staining and staining for J chain were the two most useful criteria in differentiating Ig synthesis from uptake. The results confirm that malignant B cell lymphomas synthesise monotypic Ig.


Observations on the orthograde and retrograde transport of horseradish peroxidase in the cat. Horseradish peroxidase (HRP) was applied to the precruciate cortex of the cat. The peroxidase was either applied directly to the surface or injected by glass micropipettes in single or multiple injections. Retrograde as well orthograde transport was observed by light and electron microscopy in different areas: in the thalamus: the nucleus ventralis lateralis and the nucleus centrum medianum, in the red nucleus and in the spinal cord. The distribution of HRP within the nervous elements of the nuclei: axon terminals and/or perikarya and dendrites, was observed by light and electron microscopy. HRP was present in dendrites and perikarya, indicating retrograde transport, in experiments where damage to the injected nervous tissue could be suspected. However HRP appeared restricted to the nerve endings, indicating orthograde transport, when the damage seemed to be minimal. This effect of injury on retrograde transport of HRP was further confirmed by experiments using bilateral HRP injections and unilateral lesions of the spinal cord to label rubro-spinal cells. Thus retrograde transport of HRP could be induced by lesion in systems where it does not occur normally. The normal intra-axonal transport of HRP appears to be from the perikaryon to the nerve endings. This orthograde transport can be seen under the light microscope and can be confused with faint retrograde labeling.


Augmentation of antigen-stimulated histamine release from human basophils by serum-treated zymosan particles. II. Dependence on IgE-mediated release. It was observed previously that serum-treated zymosan particles (Zx) augmented antigen and anti-IgE stimulated histamine release. With most of the enhancement attributed to an increased rate of release, this suggested that Zx was active only during the course of IgE-mediated release. This association between IgE-mediated histamine release and responsiveness to Zx was examined further in the present report. Addition of Zx at various time intervals after release had been initiated indicated that the basophil responsiveness to Zx was limited in duration; maximum responsiveness to Zx correlated closely with the period in which the rate of IgE-mediated histamine release was maximum. The time-dependent decline in sensitivity to Zx paralleled the kinetics for desensitization to antigen. Addition of Zx failed to cause release from basophils desensitized in vitro or from basophils of a donor who failed to release histamine upon challenge with anti-IgE. In contrast to the enhancement of IgE-mediated release, Zx did not augment histamine release caused by C5a or the synthetic peptide f-Met-Leu-Phe. It is concluded that an obligatory link exists between ongoing IgE-mediated histamine release and enhancement by Zx.


Adenosine receptor on human basophils: modulation of histamine release. Adenosine, at physiologic concentrations, inhibits in vitro IgE-mediated human basophil histamine release in a dose-dependent fashion. The inhibition dose-response curve is paralleled by an adenosine-induced increase in cAMP levels of human leukocyte preparations. Further evidence that the adenosine effect is related to changes in cAMP levels is that the nucleoside inhibits only in the first stage of antigen-induced histamine release and fails to inhibit the release caused by ionophore A23187. A poorly metabolized derivative of adenosine, 2-chloroadenosine inhibits as effectively as adenosine; dipyridamole, which blocks adenosine uptake, does not impair the inhibition caused by adenosine. Finally, theophylline, which is a competitive antagonist of adenosine in human lymphocytes also blocks the inhibition of release caused by adenosine. These data suggest that adenosine acts via a specific cell-surface receptor linked to adenylate cyclase. It appears that the human basophil has a specific receptor for adenosine and that this nucleoside may modulate the in vivo release of the mediators of immediate hypersensitivity reactions.


Alloantigen bound to agarose beads and syngeneic carrier cells are capable of stimulating mouse cytolytic T lymphocytes in vitro. Bead-bound antigen was prepared by coupling alloantigen covalently to agarose beads. Alloantigen-bearing syngeneic carrier cells were prepared by dilution of detergent solubilized alloantigen in the presence of syngeneic spleen cells. Both types of antigen were compared to spleen cells and reconstituted membrane fragments for the ability to stimulate cytolytic thymus-dependent lymphocytes in vitro. All these types of antigen could stimulate immune but not nonimmune spleen cells to form cytolytic T lymphocytes. The amount of lytic activity obtained with the bead-bound antigen was found to be only dependent upon the amount of H-2 antigen present in the culture and independent of the number of beads.


Non-H-2 linked control of low versus high responses of antigen-induced lymph node cell proliferation: possible role for antigen-presenting cells. Quantitative differences in the magnitude of antigen-induced proliferative responses of sensitized lymph node cells between low (C3H/Anf or C3H/Cr) and high (C3H/Hej or CBA/j) responder H-2k mice have been observed 1 to 2 weeks after in vivo sensitization with antigen (OVA, PPD, and GAT). We have shown that antigen presentation is less effective in the sensitized lymph node cell populations from low responder mice compared with those from high responder mice, suggesting that the number and/or functional status of antigen-presenting cells in the regional lymph nodes may be a key factor in determining the magnitude of antigen-induced proliferative responses. These data are consistent with the hypothesis that cell traffic after sensitization plays an important role in determining the immune responsiveness of lymph nodes.


The immune response of Lewis rats to peptide 68-88 of guinea pig myelin basic protein. I. T cell determinants. A series of peptides, produced from peptide 68-88 by selective enzyme cleavage, were used to define the amino acid sequences required for the induction of experimental allergic encephalomyelitis (EAE), an in vitro lymphocyte proliferative response (LPR), and serum antibody by peptide 68-88 in the Lewis rat. Here we present data that indicate that the T cell determinants for induction of EAE, an LPR, and helper function in the production of antibody are located in the same region of the molecule and that a minimum of 13 amino acids are involved; i.e., residues 71-85.


The immune response of Lewis rats to peptide 68-88 of guinea pig myelin basic protein. II. B cell determinants. A series of peptides, produced from peptide 68-88 by selective enzyme cleavage, were used to define the amino acid sequences reactive with Lewis rat anti-peptide 68-88 antibody. One hundred per cent of 38 rats immunized with peptide 68-88 recognized a B cell determinant residing in the region of residues 80-88. The three carboxy-terminal amino acids, residues 86-88, appeared to confer conformation on this determinant. In roughly two-thirds of the rats, the reactivity with this major B cell determinant was equal to the reactivity with the immunizing peptide. In these rats the anti-peptide 68-88 antibodies were of the IgG2 class. A third of the rats recognized an additional determinant(s) and in these the predominant class of antibody was IgG1.


A hapten-specific Ir gene.

A hapten-specific Ir gene. When mice of different strains were immunized with a conjugate of 2-phenyloxazolone (phOx) and chicken serum albumin (CSA), the antibody response was controlled by an Ir gene (Ir-phOx). H-2 alleles d and f were associated with a high response, k and a with an intermediate response, and allele b with a low response. The effect of the Ir gene was clear-cut in anti-carrier antibodies of the primary and the secondary response when the concentrations of anti-carrier antibodies varied between 1 and 350 microgram/ml. Anti-hapten antibodies reached a ceiling of ca. 1000 microgram/ml that was unaffected by the Ir gene. Before the ceiling was reached, antihapten antibodies were also subject to the control of the Ir-phOx gene. When the same carrier CSA was coupled with other haptens, BOC-ABA-Tyr or NO2phOx, antibody responses were not under the control of the Ir-phOx gene. This gene is probably responsible for the differences that have been observed earlier in delayed hypersensitivity and antibody responses to skin painting by phOx.


Studies of reticuloendothelial function in the mouse with model immune complexes. II. Serum clearance, tissue uptake, and reticuloendothelial saturation in NZB/W mice. We have extended the findings in the accompanying paper by characterizing the serum clearance and tissue uptake of model soluble immune complexes and the saturation of the reticuloendothelial system (RES) by these complexes in normal mice and in mice with murine lupus (NZB/W F1 females). Adult NZB/W or young C3H mice were injected with radiolabeled stable site-specifically cross-linked mouse anti-DNP oligomers as model immune complexes to probe RES function. Blood clearance and uptake by liver, spleen, and kidney were unimpaired in NZB/W mice. To determine if the RES exhibits partial saturation in the NZB/W mice, we deliberately induced a state of RES blockade with heat-aggregated human gamma-glubulin (HAG). With increasing doses of HAG (1 to 4 mg/20 g body weight) both strains similarly showed a progressive increase in RES saturation as measured by reduced liver uptake of model oligomers. Recovery from saturation was complete by 120 min in both strains. At maximal liver saturation there was only a small increase in oligomer uptake by kidney or spleen, the majority of complexes remaining within the circulation. Thus, RES capacity for handling soluble model immune complexes appears unimpaired in NZB/W mice.


Human blood T lymphocytes that suppress the mixed leukocyte culture reactivity of lymphocytes from HLA-B14 bearing individuals. A nontransfused male patient with recurrent bladder carcinomata has been demonstrated to have blood T lymphocytes that suppress the MLC responsiveness of lymphocytes only from normal individuals positive for HLA-B14. Unexpectedly, this T-suppressor cell differs from three other reported blood T-suppressor cells arising in man in that it apparently does not require HLA-D locus compatibility between the suppressor cell and the lymphocyte being suppressed.


Involvement of host Fc receptors in antibody-mediated cutaneous basophil hypersensitivity reactions. Cutaneous basophil hypersensitivity (CBH) reactions are heterogeneous delayed time course basophil-rich responses that can be mediated by either T cells, B cells, or serum antibodies. The current study examined the mechanism by which antibodies mediate CBH in guinea pigs. Fc competition experiments were constructed by passively transferring mixtures of anti-KLH serum and normal heterologous gamma-globulins. It was found that rabbit IgG and its isolated and purified Fc fragment [but not the (Fab')2 fragment] inhibited the ability of guinea pig immune serum to transfer CBH. Concurrent inhibition of transferred KLH-specific CBH and systemic passive cutaneous anaphylaxis (PCA) reactions by rabbit IgG or its Fc fragment, and not by sheep or bovine gamma-globulins, indicated that Fc receptors on cutaneous mast cells were probably involved in both CBH and PCA. It was also found that the basophil aspect of delayed cutaneous responses elicited by PHA was inhibited by Fc competition maneuvers. This could mean that some forms of apparently T cell-mediated CBH may be T cell dependent, but via secretion of molecules that bind to Fc receptors, as seems required in antibody-mediated CBH.


Differential activation of cytotoxic and suppressor T cells against syngeneic tumors in the mouse. The cytotoxic T cell against a methylcholanthrene-induced sarcoma, S1509a, was induced in syngeneic mice by deliberate immunization with mitomycin C (MMC)-treated live tumor cells. The soluble tumor antigen (STA) extracted from the same tumor by 3 M KCl was, however, unable to induce the cytotoxic T cell upon immunization, although it was able to activate predominantly the suppressor T cell that then specifically suppressed the effect of the cytotoxic T cell against the homologous tumor. The suppressor T cell generated by STA had the same characteristics as those found in tumor-bearing animals: 1) The suppressor T cell has a very strict specificity against individual tumors; 2) The cell expresses cell surface determinants controlled by genes in the I-J subregion of the mouse H-2 complex. The activity of the cytotoxic T cell was completely inhibited by live tumor cells but not by STA, whereas that of the suppressor T cell was neutralized by STA. The results that cytotoxic and suppressor T cells are activated under different conditions, and that the antigenic determinants recognizable by these two cell types are not the same. The soluble extract contains only the determinants recognizable by the suppressor T cell, and the cytotoxic T cell can be activated only by the determinants associated with self antigen present on the surface of live tumor cells.


M467: a murine IgA myeloma protein that binds a bacterial protein. I. Recognition of common antigenic determinants on Salmonella flagellins. We have studied the binding of M467, an IgA murine myeloma protein, to flagellin from seven species of Salmonella. It was found that M467 was reacting with antigenic determinants that were common to all the flagellins studied. These determinants were not related to serotypic antigens. Electronmicrographs of unreduced M467 showed a variety of polymeric species bound to flagella in a manner that could produce immobilization as well as agglutination and precipitation through cross-linking of antigenic determinants. Immunodiffusion in agar gel revealed that M467 was recognizing more than one group of peptide determinants on the flagellins studied. Passive hemagglutination inhibition and a solid phase radioimmunoassay provided evidence that there were differences in binding avidities between M467 and the various Salmonella flagellins studied. It was concluded that M467 is binding more than one specific group of antigenic peptide determinants on flagellin molecules. Flagellin from four of the seven species of Salmonella studied were deficient in one or more of these determinants.


Specific absorption of T lymphocytes committed to soluble protein antigens by incubation on antigen-pulsed macrophage monolayers. Monolayers of macrophages (Mphi) pulsed with antigen were used as immunosorbents for T lymphocytes from guinea pigs primed to soluble protein antigens. T lymphocytes were cultured on the Mphi monolayers for 4 hr, then aspirated and reincubated on a fresh monolayer pulsed with the same antigen for a second and a third step. T lymphocytes so treated were selectively deprived of cells responding in assay for antigen-dependent proliferation against the antigen used for pulsing the absorbing monolayer, but maintained their response to other antigens. The lymphocytes adhering to the Mphi of the absorbing monolayer were capable of giving a full response to the antigen used for pulsing the Mphi of the monolyers. The proliferative response of F1 T lymphocytes to antigen in association with Mphi of either parental strain could be absorbed leaving the response to antigen in association with Mphi of the other parental strain. The absorption of the proliferative response was not inhibited by addition of excess soluble antigen to the medium of the absorption culture. Our results indicate that specific guinea pig T lymphocytes responding by proliferation to soluble protein antigens recognize and bind specifically to a complex of Ia antigen and protein antigen at the surface of the Mphi.


Some invariant properties of IgE-mediated basophil activation and desensitization. We investigate certain general properties of antigen induced degranulation of sensitized basophils by analyzing two types of experiments: Experiments in which we expose basophils to two antigens sequentially and then determine the fraction of histamine released; and experiments in which we obtain time-dependent release and desensitization curves. To analyze the latter type of experiments we introduce a new way to plot release and desensitization data that depends on the nature of the interactions of histamine-containing units (histamine quanta) with themselves or the cells degranulation apparatus, but not on any specific properties of the antigen. From our analysis we conclude that: 1) A fraction of histamine within a population of basophils is nonreleasable by antigenic stimulation. 2) When a basophil degranulates the initial release of histamine appears to inhibit subsequent release. 3) The rate of histamine release is proportional to the amount of releasable histamine remaining in the cells when the amount remaining is small, as expected if release of histamine granules is a stochastic process. 4) There is no dependence of desensitization on the extracellular calcium concentration.


Detection of alpha-fetoprotein by supplementation counterimmunoelectrophoresis (CIE). A simple, inexpensive and sensitive method of detecting alpha-fetoprotein (AFP) by supplementation CIE assay is described. The method detects AFP at concentrations in the region of 20 ng/ml.


Characterization of in vitro proliferative responses of human lymphocytes to leishmanial antigens. Leishmaniasis is an intracellular protozoal infection for which host defense is believed to depend on cellular immune mechanisms. The in vitro proliferative responses of lymphocytes from patients with leishmaniasis and from control subjects to leishmanial antigens were examined. Only lymphocytes from patients responded to 1 microgram of leishmanial antigen/ml, whereas both patient and control lymphocytes responded to 10 micrograms/ml. The nonspecific responses were most likely not due to a mitogenic component in the antigen preparation because cord blood lymphocytes failed to respond at all concentrations of antigen tested. Both specific and nonspecific responses to leishmanial antigens were elicited in purified populations of thymus-derived (T) cells. This lymphocyte proliferation assay can be applied to the assessment of antigen-specific responsiveness of T cells from patients who represent the spectrum of host defense against leishmaniasis.


Ability of antigen-specific helper cells to effect a class-restricted increase in total Ig-secreting cells in spleens after immunization with the antigen. Immunization with antigens stimulates not only B lymphocytes secreting specific antibody but, in addition, results in the generation of very large numbers of splenic Ig-secreting cells which lack specificity for that antigen. The present report examined the nature of the antigens capable of eliciting this effect and the mechanisms whereby B cells could be nonspecifically activated. It is shown that the ability of T-dependent, but not T-independent antigens, to induce such increases requires the participation of T helper cells specific for the antigen so that any one antigen results in the activation of only a proportion of total B cells. Analysis of this nonspecific plaque-forming-cell response reveals that B cell activation is not random but occurs in a class-restricted manner. The magnitude of the increase and the isotype produced are shown to be characteristic of the immunizing antigen. Based on the data presented, the apparent nonspecific T-B collaboration can best be explained by invoking a second Ig-specific helper mechanism in which helper cells capable of recognizing determinants on Ig molecules, e.g. isotype or idiotype, cause the stimulation of B cells of any specificity providing they express that determinant.


Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. To investigate the antigenic relationship between the macrophage and lymphocyte Fc receptors (FcR), a monoclonal antibody capable of blocking mouse macrophage Fc receptors was selected. Hybrids were formed by fusing the P3U1 mouse myeloma and spleen cells from a rat immunized with the mouse macrophage-like cell lines J774 and P388D1. The Fab fragment of the monoclonal IgG secreted by clone 2.4G2, inhibited the trypsin-resistant Fc receptor II (FcRII), which is specific for immune aggregates of mouse IgG1 and IgG2b, but had no inhibitory effect on the trypsin-sensitive Fc receptor I (FcRI), which binds monomeric IgG2a and erythrocytes coated with IgG2a. Thus, the monoclonal 2.4G2 IgG appeared to be specific for macrophage FcRII. Further evidence that the 2.4G2 IgG was directed against FcRII came from binding studies of the monoclonal antibody to J774 cells and a series of independently isolated variants which do not express FcRII. These variants of J774 bound 5% as much of the monoclonal antibody as the parent line, which bound 600,000 molecules of 2.4G2 IgG per cell. The antigenic relatedness of mouse lymphocyte FcR to mouse macrophage FcRII was demonstrated by the binding of 2.4G2 IgG to FcR-bearing lymphoid cell lines and the inhibition of the lymphocyte FcR by the monoclonal antibody. Preincubation of spleen cells and peritioneal cells with 2.3G2 IgG likewise inhibited rosette formation with ox erythrocytes coated with rabbit IgG. The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex. The 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human FcR-bearing cells.


Type-specific binding antibody to baboon endogenous virus (M7) reverse transcriptase. A type-specific binding antibody to the baboon endogenous virus, M7, reverse transcriptase was developed and characterized using a double antibody immunoprecipitation assay. This assay allows the analysis of non-enzyme neutralizing binding antibody, as well as the detection of early antibody production before high titre enzyme neutralizing antibodies appear. The antibody described is unique among hyperimmune antisera to DNA polymerases in that it recognizes only type-specific determinants on the enzyme molecule. Analysis of the enzymes of several BaEV isolates indicated a grouping of those from Papio cynocephalus, P. anubis and the HL23VBab isolate. The RD-114 enzyme was in a separate class, and the P. papio and P. hamadryas DNA polymerases were distinguished from all the other BaEV enzymes but not from each other.


The strategy of infection as a criterion for phylogenetic relationships of non-coli phages morphologically similar to phage T7. Five phages which are morphologically similar to coliphage T7 but attack other host bacteria have been compared to T7 and to its relative, T3, by the following criteria: (a) cross-reactivity with antisera against T7 and T3, (b) DNA base sequence homologies, as determined by the C0t technique, (c) synthesis of two phage-coded enzymes: RNA polymerase and SAMase, (d) patterns of phage-directed protein synthesis, as determined by SDS-polyacrylamide gel electrophoresis of phage coat subunits. As judged by all these criteria, Pseudomonas phage PX3 is not related to T7; thus, morphological similarity was attributed to convergent evolution. The other phages, i.e. Serratia phage IV, Psuedomonas phage gh-1, Citrobacter phage ViIII and Klebsiella phage No. 11, were considered to be related to T7 on the basis of similarities in the patterns of phage-coded proteins and because, early after infection, these phages induced, as T7 does, an RNA polymerase which specifically transcribes the DNA of thehomologous phage. Phages IV and No. 11 also induced the early synthesis of SAMase (previously only known to occur upon T3 infection). With the exception of phage IV, however, DNA base sequence homologies with T7 or T3 seem to be poor or non-existent. The tested phages, again with the exception of phage IV, did not react with antiserum against T3 or T7. It is concluded that a particular pattern of phage-directed protein synthesis (as characterized by polyacrylamide gel electrophoresis and enzyme tests) may provide evidence for phylogenetic relationships between phages, even in cases where other criteria, such as genetic recombination, serological cross-reaction, and DNA base sequence homologies, fail to indicate relatedness.


Study of a new strain of paramyxoviruses isolated from wild ducks: antigenic and biological properties. Among the different strains of avian paramyxoviruses isolated from migrating feral ducks, two were identified as Newcastle disease viruses (NDV) and the five others would correspond to a new serotype for which we suggest the name of Duck/Mississippi/75 virus. The characteristics of this new serotype are as follows: (1) Duck/Mississippi/75 virus is able to grow as well in allantoic as in amniotic cavities of embryonated hen's eggs; (2) the haemagglutinin and haemolytic activities can be detected with hen red blood cells; (3) the neuraminidase hydrolyses the alpha 2 leads to 3 bonds of the fetuin substrate and its pH activity could be species specific. Antigenically, this serotype is different from all human and animal paramyxoviruses, in spite of an antigenic relationship with NDV.


Characteristics of a retrovirus associated with a hamster melanoma. The continuous culture of a hamster melanoma cell line has led to the spontaneous appearance of a retrovirus (HaRV) with typical type-C characteristics. The virus differs from all other known hamster viruses in its ability to transform murine as well as rat and hamster cells with apparent one-hit kinetics. Guinea pig, human and feline cells were not transformed although reverse transcriptase activity was detected in the supernatant from infected human cells. HaRV-transformed hamster embryo cells produced solid tumours (all non-pigmented) in 4 out of 35 animals when injected into hamsters while HaRV-transformed murine cells produced no tumours in mice. Injection of HaRV alone in hamsters, mice and rabbits did not induce tumours. HaRV possesses a 70S RNA which dissociates to 35S in DMSO and has a reverse transcriptase which utilizes the 70S virus RNA as a template. The size, morphology and density (1.15 g/ml) are similar to other known type-C viruses. Polyacrylamide gel electrophoresis indicates the presence of polypeptides analogous to those found in other type-C viruses.


Biochemical and immunological properties of the reverse transcriptase associated with a hamster retrovirus. Several properties of an RNA-directed DNA polymerase associated with a hamster retrovirus (HaRV) were examined and found to be similar to other polymerases from mammalian type-C viruses in that the enzyme (i) is more active with Mn2+ than Mg2+, (ii) uses the reverse transcriptase-specific poly(rCm).oligo(dG) template, (iii) possesses substantial endogenous polymerase activity and (iv) is strongly inhibited by homologous antisera and moderately inhibited by antisera directed against other type-C viruses. In contrast to previous reports of polymerases from other hamster viruses, HaRV polymerase is active in endogenous assays and the activity is associated with a 70,000 mol. wt. polypeptide in highly purified virions and with 70,000 and 85,000 mol. wt. polypeptides in fresh, unpurified virus. Only one major peak of polymerase activity eluted from DEAE-cellulose while subsequent elution of this peak from phosphocellulose produced two major peaks of polymerase activity. The mol. wt. of these two peaks were 70,000 and 85,000 by glycerol density-gradient sedimentation. The HaRV reverse transcriptase and p30 were found to be most closely related antigenically to other rodent retrovirus proteins.


Genetic recombination in Rous sarcoma virus: the genesis of recombinants and lack of evidence for linkage between pol, env and src genes in three factor crosses. Three factor crosses were performed between Rouse sarcoma virus mutants with temperature-sensitive markers in the pol and src genes and host range markers in the env gene. A number of recombinant viruses appeared to segregate from virus particles which were heterozygous for all three genes under study. The frequency of various recombinant genotypes in the progeny was consistent with there being no greater linkage between the neighbouring gene pairs of pol and env and env and src than between the more distant pol and src. The significance of these results to proposed mechanisms of avian retrovirus recombination is discussed.


Immunochemical and oligonucleotide fingerprint analyses of Venezuelan equine encephalomyelitis complex viruses. RNA oligonucleotide fingerprint analyses indicate that the genome RNA obtained from Trinidad donkey (TRD) Venezuelan equine encephalomyelitis (VEE) virus serotype I A, its vaccine strain derivative TC-83, and the VEE I B virus isolate PTF-39, have almost identical patterns of characteristic ribonuclease T1 resistant oligonucleotides. The TC-83 strain and the I B isolate can, on the basis of these analyses, be considered as variants of the TRD virus and categorized as I AB serotypes. Comparisons made by single and co-electrophoreses of the ribonuclease T1 digests of the RNA species of TC-83 and a VEE I C isolate P676 indicate that 16 of 37 large oligonucleotides of the TC-83 virus co-migrate with the oligonucleotides obtained from the I C isolate. Similar single and co-electrophoreses of ribonuclease T1 digests of the RNA species of TC-83 and a VEE I D isolate 3880 indicate that 18 of 41 TC-83 large oligonucleotides co-migrate with the oligonucleotides obtained from the I D virus isolate. At least nine of the TC-83 large oligonucleotides appear on the basis of these analyses, to be present in the digests of the genome RNA obtained from these selected I B, I C and I D virus isolates. The ribonucleast T1 digests of three I E virus isolates (Mina II, 63U2 and 71U388) give oligonucleotide fingerprints which, although comparable to each other, are more distinct from the I A and I B RNA fingerprints than are those of the I C and I D RNA species. The ribonuclease T1 resistant oligonucleotide fingerprints of VEE virus isolates belonging to serotypes (VEE subtypes) II, III and IV show little similarity to each other or to those of the serotype I virus isolates we have studied. The results obtained here agree with the reported close antigenic relationships of VEE, I A, I B, I C and I D virus isolates, and our studies suggest that these viruses have conserved nucleotide sequences. The I E virus isolates appear to have more distinct nucleotide sequences than do the other serotype 1 viruses. The results also agree with the serological differentiation of VEE, I, II, III and IV subtypes in that the oligonucleotide fingerprints of subtypes II to IV are different from each other and from those of the different serotype I virus isolates. On the basis of antigenic and genome relationships, VEE isolates can be classified as serotypes I to IV with serotype I viruses differentiated into the categories I AB, I C, I D and I E.


A radioimmunoassay for virus antibody using binding of 125I-labelled protein A. An assay for virus antibodies using protein A from Staphylococcus aureus is described. Type B and type C RNA tumour viruses adsorbed on to polystryrene microtitre plate wells were incubated with antiserum and then with 125I-labelled protein A (I-pA) and bound radioactivity was determined. Technical details such as labelling, antigen concentration, storage of I-pA are reported. The specificity of the reaction was investigated in detail by competition experiments with purified unbound homologous viruses. This assay also proved to be sensitive for demonstration of autogenous immunity to both type B and type C RNA tumour viruses. A study using antisera against purified core and envelope virus proteins of mammary tumour and leukaemis viruses suggested that the reaction mainly involves surface antigens of the intact virions.


Feline syncytium-forming virus: identification of a virion associated reverse transcriptase and electron microscopical observations of infected cells. The maturation of feline syncytium-forming virus (FSFV), a member of the foamy virus sub-family (Spumavirinae), has been studied by electron microscopy of thin sections of infected feline embryo (FEA) cells. The initial event observed was formation of crescent-shaped nucleoids at the plasma membrane. As budding progressed, the nucleoid became circular in outline with an electron-lucent centre in fully mature extracellular particles. These observations suggested that the maturation of FSFV in fully permissive FEA cells resembled that of C-type RNA tumour viruses, rather thant the B-type mouse mammary tumour virus. In this respect FSFV may be distinct from other foamy viruses. However, like other foamy viruses FSFV possessed reverse transcriptase activity. Polymerase activity co-sedimented with infectivity in an equilibrium density gradient and exhibited a preference for poly(rA).oligo(dT)10 over poly(dA).oligo(dT)10 as exogenous template.


Purification of measles virus with preservation of infectivity and antigenicity. Measles virus Edmonston strain was purified by ultrafiltration followed by two successive sedimentations through sucrose. Purified virus retained infectivity and, when used as an immunogen, elicited high titred antibody to measles antigens by conventional serology. The measles preparations were examined by SDS-PAGE followed by staining. In addition, following PAGE, the purity of these preparations was assessed immunochemically using antisera directed to measles and host cell antigens. The results of these studies demonstrate the utility of the purification method for the preparation of milligram quantities of relatively pure measles virus.


Antigens of human cytomegalovirus: electroimmunodiffusion assay and comparison among strains. The antigens of strain AD169 of human cytomegalovirus (CMV) were extracted by various methods and at different times following the appearance of cytopathic effects (c.p.e.) in infected fibroblasts. Assay with a pooled human serum in electroimmunodiffusion (EID) revealed that the most reactive preparations were obtained by shell-freeze (SF) extraction on the fourth day after 4+c.p.e. As many as 20 antigens could be detected in the original gels, most of which were stable upon storage at 4 degrees C for up to 4 weeks; of these, about 14 can be reproducibly seen on photographs. EID runs on day 4 SF preparations from high-passage CMV strains C87 and Davis and low passage recent isolates VD14, 1694 and 1723 resolved, respectively, 15, 15, 13, 11 and 11 antigens in the original gels (11, 9, 11,8 and 9 are visible in photographs). Strains 1694 and 1723 shared fewer antigens with one another and with high passage strains than were shared among the latter, whereas VD14 had relatively large numbers of antigens common to both low and high passage strains. At least six antigens were common to all strains.


Antigenic relationships between polypeptides derived from plantar and hand wart viruses. Guinea pigs immunized with polypeptides derived from plantar and hand wart viruses developed both humoral and cell mediated immunity. The specificity of the antiserum was determined by indirect immunofluorescence (IF) tests and cellular immunity by intradermal tests. While whole plantar and hand wart virus particles (PV and HV) appeared to be immunologically analogous, they had different polypeptide patterns as shown by analysis on acrylamide slab gels. In particular, the P2 polypeptide (major virus protein) with different mol. wt. for PV (56750) and HV (54500) induced the production of high antibody titres in the animals and the immune sera specifically labelled wart substrates as shown in thie IF test, demonstrating that no cross humoral reaction occurs between these two polypeptides. Furthermore, a delayed hypersensitivity reaction was observed in P2 polypeptide-inoculated guinea pigs when whole particles were introduced in skin tests, but a total cross reactivity between PV and HV was noticed at the cellular level. However, the study of the virus isolated from the lesions of a patient (Ri) bearing extensive common hand warts has shown that the virus particles possessed all the biochemical and immunological characteristics of PV, in particular with regard to the P2 polypeptide. Such a case may represent plantar-like warts located on the hands.


Cross-reactive and type-specific complement-fixing structures of Oriboca virions. Characteristics of two components of Oriboca virus were studied after they were separated and semipurified by velocity centrifugation. A component of low infectivity and broad cross-reactivity in the complement-fixation (CF) test had a sedimentation coefficient of 6--7S. Infectious Oriboca virions with hemagglutinating (HA) activity had a sedimentation coeffecient of 457S. These virions cross-reacted broadly with Murutucu viral antibody. The cross-reactive, virion-associated component and a type-specific CF fragment were released from virions by disruption with Nonidet P-40. These CF fragments separated by sucrose gradient equilibrium centrifugation had densities of 1.245gm/ml and 1.181 gm/ml, respectively, and were both interpreted to be envelope structures. The type-specific CF antigen appeared to be related to the viral hemagglutinin in that it blocked hemagglutination-inhibiting (HI) antibody, whereas the cross-reactive CF fragment did not.


Aspects of cellular immunity in multiple sclerosis. Antigen-reactivity of lymphocytes and lymphokine activity. Some new results of cell-mediated immunity in multiple slcerosis (MS) are presented, based on the determination of charge-changing lymphokines as products of antigen sensitive lymphocytes (C PAL), obtained by several forms of the electrophoretic mobility (EM) method. Lymphocytes from MS react to myelin basic protein (BP), the reactivity in other neurological diseases depending on the degree of destruction of nervous parenchyma. Applying a membrane-associated antigen from normal brain (NTA), positive reactivity of MS lymphocytes was obtained. Besides the usual determination of lymphokines in vitro the sensitive EM test allows the demonstration of lymphokine activities in vivo; i.e. in body fluids such as cerebrospinal fluid (CSF) and serum. In CSF of MS a high lymphokine activity was found. The differentiation of lymphokines and comparison between in vitro and in vivo activity were carried out. Moreover, a characteristic lymphokine pattern for MS with high activities in all regions of molecular weight, especially in the CSF, could be detected. On the basis of these findings, important also from the pathogenetic point of view, a diagnostic scheme for MS is suggested, consisting of a program of determination of the immuno-reactive CSF syndrome and some special procedures, including examination of lymphocyte reactivity.


Delayed hypersensitivity to human encephalitogenic protein as assayed by agarose leucocyte migration in multiple sclerosis patients. Using a leucocyte migration test (Clausen's direct agarose gel migration method) hypersensitivity to human encephalitogenic protein has been examined in 50 multiple sclerosis patients (group 1), 50 healthy persons (group 2) and 25 patients with other neurological diseases (group 3). In group 1, 30 MS patients (60%) show an abnormal migration index, manifested either as inhibition or stimulation of migration; 29 controls in group 2 (58%), 11 O.N.D. patients in group 3 (44%) show an abnormal migration index. These results mean that lymphocyte hypersensitivity to myelin basic protein appears neither to be constant nor specific to multiple sclerosis. Three migration index curve types at different antigen concentration are obtained: monophasic curves within the normal index zones; monophasic curves staying in the inhibition or stimulation zone and biphasic curves with dose-effect relationship. Whatever the antigen used, this dose-effect relationship implies that the test must be carried out at different concentrations. The meaning of spontaneous sensitisation in healthy controls is discussed.


E-PTA stains oligodendroglial surface membranes and microtubules in optic nerves during myelination. Aldehyde fixed Xenopus tadpole and frog optic nerves were stained en bloc with ethanolic phosphotungstic acid (E-PTA). During rapid myelination, intense staining was observed on cytoplasmic faces of paranodal terminal loops and loosely wrapped oligodendroglial membranes found along inner and outer surfaces of compact myelin sheaths. Oligodendroglial microtubules also were heavily stained. Where stained cytoplasmic faces fused to form a lamella of compact myelin, the intense staining was reduced to a thinner, fainter line. In optic nerves of adult frogs, the staining was less dense but the pattern was similar. The staining distribution and available histochemical evidence indicate that E-PTA stains positively charged proteins non specifically. Since myelin basic protein is found in oligodendroglia during myelination, we suggest that it is being stained by E-PTA while being transported along microtubules to sites where it is inserted into developing myelin lamellae.


Proteinase inhibitors in cerebrospinal fluid in multiple sclerosis. Levels of the proteinase inhibitors alpha 2-macroglobulin (alpha 2-m) and alpha 1-antitrypsin (alpha 1-at), and total protein, IgG and transferrin were measured in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurological diseases. All groups except that termed "meningitis" had similar alpha 2-m levels, but alpha 1-at and transferrin were significantly depressed in MS. Total protein levels were normal and IgG levels were elevated in MS. Serum levels of alpha 1-at were normal so the decreases observed in the CSF in MS were not due to impaired systemic production. In view of previous reports that proteinase activity is high in MS plaques and CSF, the inhibitory capacity of alpha 2-m and alpha 1-at in CSF was measured. As any decreases in inhibitory capacity noted in MS were slight, they could only be important in the local environment of a plaque where enzyme levels may be critically high.


The effect of Cop 1, a synthetic polypeptide, on chronic relapsing experimental allergic encephalomyelitis in guinea pigs. Cop 1, a synthetic polypeptide, was evaluated for its effect on a chronic relapsing form of experimental allergic encephalomyelitis (EAE). Pretreatment of juvenile Strain 13 guinea pigs with Cop 1 in incomplete Freund's adjuvant (IFA) which were subsequently challenged with guinea pig spinal cord in complete Freund's adjuvant (CFA) had a marked effect in delaying or preventing the appearance of clinical signs of EAE. Administration of Cop 1 on appearance of clinical signs of EAE prevented progression of the first episode of the disease. Although relapses were not always prevented, they were modified on their duration and intensity both clinically and histologically.


Direct immunofixation after isoelectric focusing. An improved method for identification of cerebrospinal fluid and serum proteins. An improved method for identification of CSF and serum proteins is described, using analytical isoelectric focusing followed by direct immunofixation in polyacrylamide gel. This method offers high sensitivity together with retained resolution after isoelectric focusing and is technically easy to perform. The gamma-trace protein, normal transferrin, haptoglobin and alpha 2-macroglobulin and two genetic alterations of transferrin were analyzed. The qualitative differences, previously observed on crossed immunoelectrofocusing, between transferrin and alpha 2-macroglobulin in the CSF and serum were confirmed by immunofixation. Protein components, differing in isoelectric pH by at least 0.03 pH unit and 0.5 mm apart were readily identified by this technique.


Rhythmic palatal myoclonus and the dentato-olivary pathway. The anatomical basis of palatal myoclonus and related rhythmic skeletal myoclonus is described. The most constant lesion is a special type of degeneration with hypertrophy of the olivary nucleus of the medulla oblongata, on the side opposite to the myoclonus when it is unilateral. This degeneration is usually secondary to a primary lesion located either in the ipsilateral (to the hypertrophied olive) central tegmental tract or in the contralateral dentate nucleus. To link these data, Trelles (1935, 1943) suggested a dentato-olivary pathway from the dentate nucleus to the contralateral inferior olive via the superior cerebellar peduncle and the central tegmental tract after crossing the midline. The existence of this pathway was demonstrated by Lapresle and Ben Hamida (1965-1971), first by showing a topistic relationship between dentate nucleus and contralateral inferior olive, then by delineating this pathway in the vicinity of the red nucleus at the crossing of the superior cerebellar peduncle and the central tegmental tract. The significance of these lesions with their ensuing symptoms is discussed. It is considered as a transsynaptic degeneration which probably reveals an archaic phenomenon, submerged but not lost through evolution.


Neuromyopathy during chronic amiodarone treatment. A case report. Clinical, electrophysiological, and the nerve and muscle biopsy findings from a case treated with amiodarone are reported. Marked distal motor and sensory impairment and distal muscular atrophy were observed clinically. The electrophysiological examination revealed normal motor and sensory conduction velocities in the median nerve; the sensory action  potentials were polyphasic and reduced in amplitude. Electromyography revealed denervation potentials and severe loss of motor units in the M. extensor digitorum brevis and in the M. tibialis anterior. The light and electronmicroscopical study of a N. suralis biopsy displayed total loss of large myelinated fibers and an almost total reduction of small myelinated fibers. The number of unmyelinated axons was markedly reduced. Fibrocytes and degenerative axons polymorphous inclusion bodies were present in Schwann cells. The muscle biopsy revealed both neurogenic and myopathic changes. Lipid storage was also present in the muscle fibers. Physical and chemical analysis of the nerve and muscle biopsy revealed the content of iodine to be more than 40 times increased. The findings indicate damage of axons, schwann cells and muscle fibers. It is suggested that the lipid storage in nerve and muscle tissue might be related to the accumulation of the drug or its metabolites.


Clinical and ultrastructural study of a sporadic case of hereditary sensory neuropathy. Morphological evidence for a slow rate fiber degeneration. A sporadic case of hereditary sensory neuropathy, with a clinical course and severe trophic and sensory alterations typical for type II, is presented. There was a severe loss of myelin in the sural nerve biopsy taken from the ankle. The most impressive microscopic feature was the number of rudimentary onion bulbs with an empty core which contained nude axons in the peripheral schwann cell layers; they were interpreted as vestigial structures left by the former myelinated fibers. Electron microscopy also revealed a definite involvement of unmyelinated fibers with attempted regeneration, which was confirmed by the overrepresentation of small axons on their frequency distribution curve. The whole ultrastructural picture suggested the protracted nature of the fiber involvement. This may be considered as agreeing with the slow course proposed for a system degeneration.


Paramyotonia congenita. A clinical, electrophysiological and histological study of 12 patients. The present paper describes the clinical, electrophysiological and histological findings made in 12 patients belonging to 2 genealogical lineages in Switzerland, suffering from paramyotonia congenita. This is the first report of this disease in Switzerland. The myopathy, transmitted by autosomal dominant inheritance, is characterized by a typical past medical history and by the persistent contraction of the muscles of the face, arms and legs provoked by exposure to cold. Also of diagnostic importance are the myotonic reactions inducible at room temperature, such as percussion myotonia, active myotonia and paradoxical myotonia. During paramyotonic episodes provoked by exposure of the subjects in a refrigeration chamber, serum potassium concentrations remained within normal limits. Potassium loading producing serum levels above 6 mEq/1 in 2 patients gave rise to stiffness and weakness of the arms and legs, but no signs of paralysis. Induced hypokalemia in 3 cases caused no paramyotonic symptoms. Electromyographic recordings in 5 patients showed myotonic discharges, which disappeared upon cooling of the limb, giving way to progressive muscular stiffness. Histological, histochemical and electronmicroscopical examination of the muscle tissue revealed only diagnostically unspecific myopathological changes.


Primary malignant lymphoma of the CNS and polyneuropathy in a patient with necrotizing vasculitis treated with immunosuppression. A woman, aged 58, who had undergone prolonged treatment with corticosteroids for generalized necrotizing vasculitis, and had received azathioprine (Imurel) for 18 months, 4 years after discontinuing the latter treatment, developed peripheral neuropathy and a rapidly progressing cerebral disorder suggesting a basal meningeal process. CSF cytology suggested malignant lymphoma with meningeal involvement. Immunological studies showed an increase of Null-lymphocytes in the peripheral blood. Autopsy disclosed a primary malignant lymphoma of the CNS with the histological appearance of a multilocular immunoblastoma showing almost ubiquitous meningocerebral involvement. Clinical and postmortem examinations failed to demonstrate any systemic extraneural lymphoproliferative disorder. In addition, there was peripheral polyneuropathy of the axonal type with denervation atrophy of skeletal muscle, but without lymphomatous involvement of the neuromuscular system. The possible relations between primary malignant lymphomas of the CNS and previous immunosuppressive treatment of immunoinflammatory disease are discussed in view of the concept of impaired immunoregulation. The pathogenetic background of peripheral polyneuropathy is unknown.


Infantile cortical measles inclusion body encephalitis during combined treatment of acute lymphoblastic leukemia. A case of cortical measles inclusion body encephalitis occuring in a boy aged 6 years 7 months, 4 months after uncomplicated measles is reported. The child was undergoing combined treatment ofr acute lymphoblastic leukemia. He was in primary remission for 2 years. The neuropathological findings are characterized by necrosis, eosinophilic nuclear and cytoplasmic inclusion bodies in the neuronal and glial cells within the cerebral cortex and basal ganglia. Nucleocapsides of paramyxoviruses were detected in the nuclear inclusion bodies of both cell types.


Cytostatic treatment of meningeal blastoses. Intrathecal treatment with MTX was attempted in patients with diffuse meningeal blastoses. As a rule, symptoms and clinical findings improved rapidly. As objective parameters, the differential cell pictures of the CSF showed dissimilar results during therapy (25 mg MTX weekly i.t.). In leukoses (n = 10) and malignant lymphomas (n = 12), the CSF could be cleared with three exceptions which concerned differentiated tumors. The success was similarly good with meningeal dissemination of a seminoma. Meningeal carcinoses with mammary carcinoma (n = 7) and melanoma (n = 2) showed dissimilar results. No alteration of the cytograms of four bronchial carcinomas and one colon carcinoma was demonstrable. Besides the growth form, the degree of differentiation appears to be decisive for the success of therapy. The labeling index appears to be a useful indicator. Since symptoms and clinical findings often markedly improve even when a success cannot be objectively detected cytologically, an attempt at therapy is always to be recommended.


Urinary glycosaminoglycans in patients with progressive myoclonus epilepsy. Urinary GAGs analysis in Progressive Myoclonus Epilepsy (PME) showed an accumulation of uronic acid in the fraction eluted by 1 M NaCl and 3 M NaCl. As analogous changes were found in other myoclonic and epileptic patients receiving large doses of anticonvulsant drugs, these alterations in the GAG urinary pattern were not considered a primary disturbance of PME.


Immunodeficiency in epilepsy: a new view. Up to 12% of epileptic patients have subnormal IgA serum concentrations. Previous observations suggest that IgA deficiency is correlated with hydantoin treatment and also with the type of seizure. In a followup study it is shown that IgA deficiency in epileptics is a rather constant feature of a given patient. The most pronounced changes in IgA levels were seen in patients in whom the hydantoin medication also changed. However, low IgA levels have also been reported in untreated epileptics. A new classification for the immunodeficiency state in epileptics is introduced.


Rapid detection of malaria and other bloodstream parasites by fluorescence microscopy with 4'6 diamidino-2-phenylindole (DAPI). DAPI is a fluorescent dye which appears to complex specifically with DNA. We have used this probe to detect and identify malarial infections by fluorescence microscopy. Experiments were conducted using Plasmodium berghei yoeli--infected mouse blood, P. lophurae--infected duck blood, and P. vivax--infected human blood. Infected avian blood was used to detect parasites within nucleated erythrocytes. Control blood smears from uninfected hosts revealed fluorescence only in the leukocytes of mammalian blood or in nuclei of leukocytes and erythrocytes of avian blood. Cytoplasmic staining of red blood cells was absent in all controls. In contrast, the cytoplasm of infected red blood cells was stippled with fluorescence centers. Ring forms, trophozoites, segmenters, and merozoites frequently were observed. This simple procedure can be applied directly to routine clinical analysis, as well as experimental procedures, DAPI can also be used to stain other parasites, including nuclei in microfilariae.


Inhibition of fertility in mice by passive immunization with antibodies to isolated zonae pellucidae. A chromatographically purified preparation of gamma globulin produced against isolated zonae pellucidae of mice was used to immunize mice. A single 2.5 mg dose totally inhibited fertility for a minimum of 11 days.


Treatment of a retarded child's faeces smearing and coprophagic behaviour. The daily rearrangement of a profoundly retarded child's routine shower was effective in eliminating his low-frequency faeces smearing and coprophagic behaviour during the late afternoon/early evening part of the day. The effects appeared to generalise such that both types of behaviour were reduced at other times of the day and night. These results are discussed in terms of a social learning framework. A model for analysing the motivations and reinforcing conditions for rectal digging, coprophagy, and smearing faeces is also presented.


The evaluation of cell nuclei staining parameters regarding the reproducibility of fluorescence measurements. The present methodological study is an attempt to optimize the staining parameters for a quantitative DNA determination of fluorescent cells. The application of pure dyes and a precise control of the staining procedure are preliminary conditions which have to be fulfilled, because the reproducibility of measurements is in this connection the most important criterion for a quantitative DNA related analysis of cell population. Chicken erythrocytes and isolated nuclei were applied as biological test objects. The staining procedure with acridine derivatives (acriflavine, proflavine, rivanol) was performed in accordance with the fluorescence-Feulgen reaction. The influence of staining parameters, such as (1) pH and (2) dye concentration of the staining solution, were evaluated regarding the spectral behaviour, the total fluorescence intensity, and the reproducibility of results.


Depression of DNA synthesis in mouse spleen after treatment with 5-aza-2'-deoxycytidine. 5-Aza-2'-deoxycytidine is a highly effective cytostatic agent that preferentially affects the lymphatic system. Pretreatment of noninbred H mice with the drug markedly depressed the level of thymidine (dThd) incorporation into DNA in the spleen and also lowered the dThd and thymidylate kinase activities. Maximum effects were observed following administration of the analog in a single dose 24 hours before the mice were killed. Whereas cytidine and dThd did not reverse the inhibitory effect of 5-aza-2'-deoxycytidine, excessive doses of deoxycytidine partially reversed this inhibition. Similar to the depression of dThd incorporation, a depression in the incorporation of deoxycytidine and cytidine into spleen DNA was found after 24-hour pretreatment with 5-aza-2'-deoxycytidine. However, 7 days following 5-aza-2'-deoxycytidine treatment, the incorporation of dThd into DNA in the spleens of mice was significantly increased. [3H]5-aza-2'-deoxycytidine was rapidly incorporated into spleen DNA, whereas deoxycytidine interfered with the incorporation of [3H]5-aza-2'-deoxycytidine.


Differential growth inhibition in two human carcinoma cell lines by cyclic adenosine 5'-monophosphate analogs. The inhibition of cell replication in two human carcinoma cell lines by various cyclic AMP analogs was explored. In all instances, the addition of the cyclic nucleotide phosphodiesterase inhibitor 1-methyl-3-isoburylxanthine resulted in synergistic growth inhibition by the analogs. A correlation was found between an analog's ability to inhibit growth and its ability to activate protein kinase. A differential effect of the breakdown product 8-bromo-AMP (8-BrAMP) on cell replication in the two cell lines was observed; i.e., one cell type was extremely sensitive to inhibition by 8-BrAMP and the growth inhibition could not be reversed by uridine, whereas the other cell type was less sensitive to 8-BrAMP and the growth inhibition was significantly reversed by uridine.


Isolation of an endogenous type C virus related to the infectious primate type C viruses from the Asian rodent Vandeleuria oleracea. A tissue culture line derived from the Asian rodent Vandeleuria oleracea has been shown to release an infectious, xenotropic type C virus. The virus-associated reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and the major internal protein p30 are immunologically related to the respective proteins of the woolly monkey-gibbon ape group of infectious primate viruses. By these criteria the V. oleracea viral isolate is similar to the murine type C-I class of endogenous retroviruses and has been designated Vand C-I. Nucleic acid homology studies show that V. oleracea cellular DNA shares similar levels of homology with DNA from members of the Mus and Rattus genera and lower levels of homology with other rodent genera. The Vand C-I viral genome is present in V. oleracea cellular DNA in multiple copies, and partially related sequences can be detected in other rodent genera. These results support the conclusion that the Vand C-I viral genome is genetically transmitted in V. oleracea and that the type C-I class of endogenous retroviral genes has been highly conserved during evolution.


Immunological characterization of mouse mammary tumor virus p10 and its presence in mammary tumors and sera of tumor-bearing mice. Mouse mammary tumor virus (MMTV) p10 and gp52 were purified and used as radiolabeled antigens in sensitive radioimmunoassays. These radioimmunoassays were specific for MMTV proteins since detergent-disrupted MMTV from C3H/HeN, RIII, and GR/N mice gave complete competition, whereas C3H/HeNf liver extracts and other lysed retroviruses did not. Both gp52 and p10 are coded by the viral genome, since MMTV grown in a heterologous cell line (feline kidney cells) competed in these assays. Sera from mammary tumor-bearing mice and mammary tumors from C3H/HeN and C3H/HeNf mice competed in both the gp52 and the p10 assays. Although these radioimmunoassays detected predominantly group-specific antigenic determinants in C3H/HeN and C3H/HeNf tumor extracts, type specificity was also found with gp52. Absorption of the anti-MMTV serum with C3H/HeNf tumor extracts removed all antibodies directed against p10 and decreased the anti-gp52 titer approximately 30-fold. When this absorbed antiserum was used at limiting dilution in the gp52 radioimmunoassay, C3H/HeN tumor extracts gave complete competition, whereas no competition was found with C3H/HeNf tumor extracts.


Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells. We have studied the structure of polyadenylated virus-specific cytoplasmic mRNA's in mouse and human cells transformed by simian virus 40 and in monkey cells infected with simian virus 40 in the presence of cytosine arabinoside by means of reverse transcriptase-catalyzed complementary DNA synthesis and complementary DNA sequencing. Abundant mRNA species containing splices from residues 4490 to 4557 (0.533 to 0.546 map units [m.u.]) and 4490 to 4837 (0.533 to 0.600 m.u.) were identified in both transformed and infected cells. Two principal reverse transcriptase stops were observed at the 5' termini of these mRNA's, both occurring with approximately equal frequency. The most distal of these stops was localized at residues 5152 to 5154 (0.660 m.u.), and the second was at residues 5147 to 5148 (0.659 m.u.). Several additional minor stops, between approximately 0.62 and 0.65 m.u., were also found on complementary DNA copied from transformed cell mRNA; in contrast, only one additional stop was present on complementary DNA copied from early lytic mRNA. These data suggest the presence of a prinicipal 5' terminus of early lytic and transformed cell mRNA's at residues 5152 to 5154 and raise the possibility of additional 5' termini at one or more locations in the 0.62 to 0.659 m.u. region of these mRNA's. Transformed cell mRNA was also found to contain a single 3' terminus at positions 2504 and 2505 (0.153 m.u.); termini lying beyond this site were not detected.


Initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro: nature and location of the oligodeoxycytidylic acid primer binding site. We have investigated the use of oligodeoxycytidylic acid [oligo(dC)] as a primer for the initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro, employing the viral RNA genome as template. The addition of oligo(dC)(12-18) to viral 35S RNA results in a stimulation of DNA synthesis by the viral RNA-directed DNA polymerase comparable to that observed when oligo(dT) is employed as a primer. Under similar conditions neither oligo(dA)(12-18) nor oligo(dG)(12-18) was active as primer for transcription of the avian retrovirus genome. Several different approaches have been employed to localize the oligo(dC)(12-18) binding site on the viral genome, including isolation of poly(A)-containing fragments, competition hybridization, and RNase H hydrolysis. These analyses indicate that oligo(dC)(12-18) binds to a site approximately 2,000 to 3,000 nucleotides from the 3' terminus of the genome of transforming strains of avian sarcoma viruses and approximately 700 to 1,000 nucleotides from the 3' terminus of nontransforming avian retroviruses. Therefore, the major site of initiation of DNA synthesis by oligo(dC)(12-18) appears to be in the vicinity of the 3' end of the env gene and the 5' end of the src gene, although the presence of minor initiation sites located elsewhere on the viral genome cannot be excluded by these data. Characterization of oligonucleotides after pancreatic RNase hydrolysis and poly(C)-Sepharose chromatography of viral RNA directly demonstrates the presence of oligoguanylic acid residues in the avian sarcoma virus genome. DNA sequences transcribed from the oligo(dC) primer appear to be conserved in all of the avian leukosis-sarcoma viruses tested. The use of oligo(dC) as a tool for the production of specific complementary DNA probes is discussed.


Adsorption, penetration, and uncoating of murine leukemia virus studied by using its reverse transcriptase. A procedure using the virus-associated reverse transcriptase was developed for following the kinetics of adsorption, penetration, and uncoating of murine leukemia virus. Viral adsorption to cell membrane was determined by assaying this enzyme activity in isolated debris of mechanically disrupted cells after infection with murine leukemia virus in the presence of actinomycin D. At 37 degrees C, viral adsorption proceeded at a high initial rate, but after 5 min of incubation with the virus, it gradually slowed down. At 4 degrees C, viral adsorption was slower but proceeded at a linear rate. Intracellular virus was determined by centrifuging the cytoplasmic fraction of the disrupted cells at 105,000 x g for 45 min and assaying reverse-transcriptase activity in the high-speed pellet thus obtained. Sucrose gradient analysis of the enzyme activity recovered from the cytoplasm of infected cells indicated that this activity represented intact virus particles. No appreciable amount of such particles was recovered from the cytoplasm of cells infected at 4 degrees C. This indicates that the virions recovered from the cytoplasm of cells infected at 37 degrees C are indeed intracellular virus particles which penetrated into the cells and not just membrane-bound particles mechanically released to the cytoplasmic fraction during cell disruption. By this procedure intracellular virus was found to accumulate in the cytoplasm, reaching a maximal level within 20 min. The accumulated intracellular virus particles gradually disappeared from the cytoplasm, evidently due to their uncoating which was completed within 80 min.


Biochemical characterization of the type C retrovirus associated with lymphoproliferative disease of turkeys. Turkeys inoculated with spleen extracts from lymphoproliferative disease (LPD)-affected birds developed viremia, followed by typical LPD lesions. Electron microscopy and biochemical characterization established that the virus present in the blood of infected turkeys is a type C retrovirus. The viral particles possess a buoyant density of 1.17 g/ml in sucrose gradients; they contain high-molecular-weight RNA and an RNA-instructed DNA polymerase with efficient exogenous and endogenous activity. The LPD virus polymerase is preferentially activated by magnesium ions. Cross nucleic acid hybridization assays revealed no sequence homology between the viral genome of LPD and avian myeloblastosis virus or reticuloendotheliosis virus, thus indicating that the LPD virus belongs to a distinct group unrelated to the avian leukosis-sarcoma virus complex or to the reticuloendotheliosis virus group.


Structure of products of the Moloney murine leukemia virus endogenous DNA polymerase reaction. We have investigated the process by which the single-stranded RNA genome of Moloney murine leukemia virus is copied into DNA in vitro. DNA synthesis if initiated near the 5' end of the genome, and the elongation of the growing chain occurs by a jumping mechanism whereby the DNA synthesized at the 5' end of the genome is elongated along the 3' end. Unique DNA fragments synthesized beyond the 5' end of the genome in vitro have, at their 5' and 3' ends, copies of unique sequences from the 5' and 3' ends of the genome. These flank a copy of the 49- to 60-nucleotide terminally redundant sequence. These results indicate that the terminal redundancy serves as a "bridge" to allow a DNA molecule synthesized at the 5' end of the genome to serve as a primer for synthesis from the 3' end.


Expression of baboon endogenous virus in exogenously infected baboon cells. Strains of low-passage, fetal diploid, baboon (Papio cynocephalus) fibroblasts were susceptible to exogenous infection with three independent isolates of baboon endogenous virus, as measured by an immunofluorescence assay specific for viral p28. Infectivity of the M7 strain of baboon endogenous virus for baboon cells of fetal skin muscle origin was equivalent to that for human and dog cells in that similar, linear, single-hit titration patterns were obtained. The assay for supernatant RNA-dependent DNA polymerase, however, showed that baboon cells produced only low levels of virus after infection compared with the production by heterologous cells. The results showed that baboon endogenous virus was capable of penetrating baboon cells and that viral genes were expressed in infected cells. Replication of complete infectious virus was restricted, however, indicating that in this primate system homologous cells differentially regulated the expression of viral genes.


Identification of the primate papovavirus HD as the stump-tailed macaque virus. The recently isolated primate papovavirus HD is shown to be indistinguishable from the stump-tailed macaque virus by immunofluorescent reactivity, by restriction endonuclease analysis, and by nucleic acid hybridization assay.


Analysis of proteins of mouse sarcoma pseudotype viruses: type-specific radioimmunoassay for ecotropic virus p30's. Murine sarcoma virus pseudotypes were prepared by infection of nonproducer cells (A1-2), which were transformed by the Gazdar strain of mouse sarcoma virus, with Gross (N-tropic), WN1802B (B-tropic), or Moloney (NB-tropic) viruses. The respective host range pseudotype sarcoma viruses were defined by the titration characteristics on cells with the appropriate Fv-1 genotype. Proteins from virus progeny were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bands present in both the 65,000- and the 10,000- to 20,000- molecular-weight regions of the gel distinguished the pseudotype viruses from their respective helpers. Furthermore, two protein bands were noted in the p30 region of murine sarcoma virus (Gross), one corresponding to Gross virus p30, and another of slightly slower mobility. However, since the mobility of the putative sarcoma p30 is nearly indentical to that of WN1802B, its presence could not be established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Type-specific radioimmunoassays for Gross virus p30 and for WN1802B p30 were applied for analysis of pseudotype preparations, and among several ecotropic viruses tested, only the homologous virus scored in the respective assay. By use of these assays, pseudotype viruses were found to contain only 8 to 48% helper-specific p30's; the remainder is presumably derived from the sarcoma virus.


Use of UV irradiation to identify the genetic information of vesicular stomatitis virus responsible for shutting off cellular RNA synthesis. UV irradiation of infectious vesicular stomatitis virus was employed to study the relationship between the expression of certain viral gene functions and viral inhibition of RNA synthesis in mouse myeloma (MPC-11) cells. Viral infectivity, protein synthesis, and viral mRNA synthesis were all highly susceptible to inactivation by UV radiation; however, low levels of viral transcriptase activity were detected in vitro in virus preparations subjected to large doses of UV radiation.  In sharp contrast, the capacity of vesicular stomatitis virus to shut off cellular transcription was quite resistant to UV radiation. The data presented here indicate that viral transcription is essential to inhibit host RNA metabolism, even though synthesis of viral polypeptides in the inhibited cells could not be detected. At those levels of UV radiation that inactivated all viral gene functions, except viral inhibition of cellular RNA synthesis, the only viral product detected was non-adenylated, low-molecular-weight RNA species.