Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos. I. Toluidine blue and lanthanum staining. Four developmental stages of Rana pipiens, R. pipiens female X R. catesbeiana female, and R. pipiens female X R. esculenta male embryos were fixed in standard electron microscopic fixatives with or without 1% lanthanum nitrate (LN) added. Embryos fixed without LN were embedded and sectioned at 0-5 micrometer and stained with toluidine blue. Embryos fixed with LN were embedded and sectioned at 70-80 nm and examined in the electron microscope. Both techiques show a dramatic increase in the amount of stainable material in the extracellular matrix during development. Blastula stage embryos have a small amount of material in the extracellular matrix. Early gastrulae have more stainable material than blastulae and this increased amount appears to be due to an accumulation of material in the marginal zone of the embryo, especially near the dorsal lip of the blastopore. Late gastrulae have large amounts  of stainable material in the extracellular spaces in almost all parts of the embryo. Interspecific arrested hybrid embryos do not show the same dramatic accumulation of stainable material in their extracellular matrices.


Karyotypic analyses of parental and hybrid intraspecific mouse cells, A9/B82, by giemsa- and centromeric-banding. Hybrids between A9 (HGPRT-) and B82 (TK-), mouse heteroploid fibroblast lines, were obtained through continuous cultivation and clonal selection; such hybrids showed marked segregation and by conventional stains displayed chromosome numbers and distribution similar to that of either parental type. Detailed analyses by Giemsa (G)- and centromeric-banding of these parental lines, and of 4 of the reduced hybrids, maintained in culture for up to 5 years, revealed the following points: (1) The distribution of the majority of individual chromosomal classes was similar for 3 of the hybrid cell lines. (2) Over two-thirds of the chromosomal arms in both the parental lines and hybrid lines were identical to normal mouse telocentric chromosomes. (3) For 2 of the hybrid lines, segregation was particularly marked with respect to those chromosomal arms whose G-banding patterns were identical to the wild-type genome; this indicated that segregation had occurred at the expense of redundant chromosomal material introduced by cell fusion. These banded studies demonstrated that segregation chiefly accounted for the sharp reduction in chromosome numbers while recombination accounted for the chromosome heterogeneity of the hybrid cells as compared to the parental genomes.


Platelet membrane defects in Glanzmann's thrombasthenia. Evidence for decreased amounts of two major glycoproteins. Platelets from patients with Glanzmann's thrombasthenia have a distinct molecular alteration of the plasma membrane surface, namely decreased amounts of a major glycoprotein designated as IIb (apparent mol wt 142,000). To identify other possible surface defects of thrombasthenic platelets, we labeled the membrane polypeptides of normal and thrombasthenic platelets by two different techniques: lactoperoxidase-catalyzed iodination and galactose oxidase oxidation, followed by reduction with tritiated sodium borohydride. Labeling patterns were determined after the polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Before the second dimension was run, platelet samples were incubated with a reducing agent, beta-mercapto-ethanol, to cleave the disulfide bonds of certain glycoproteins; the resulting changes in electrophoretic mobility permitted better resolution of individual molecules. Comparison of the labeled polypeptides of normal and thrombasthenic samples after reduction indicated decreased labeling of two major glycoproteins in thrombasthenic platelets: IIb and III (apparent mol wt 114,000). The relative proportions of radioactivity incorporated by these polypeptides were about 60 and 80% less than control values, respectively. With either Coomassie Blue or periodic acid-Schiff's reagent, glycoprotein III stained much less intensely in thrombasthenic compared to normal samples, indicating that the observed labeling deficit was caused by a decreased concentration of the molecule rather than steric inaccessibility on the membrane surface. Analysis of normal plasma membranes by affinity chromatography showed that glycoprotein IIb has receptors for lectin from Lens culinaris, the common lentil, whereas III does not. We conclude that a characteristic feature of Glanzmann's thrombasthenia is a decreased concentration of two discrete glycoproteins in the platelet plasma membrane.


Synthesis of factor VIII antigen by cultured guinea pig megakaryocytes. Immunoprecipitates containing guinea pig Factor VIII antigen were prepared from guinea pig plasma with a cross-reacting rabbit anti-human Factor VIII. Monospecific antisera to guinea pig Factor VIII antigen were produced in rabbits by using these washed immunoprecipitates as immunogens. The resulting antisera to guinea pig Factor VIII antigen detected Factor VIII antigen in guinea pig plasma and inhibited the von Willebrand factor activity in guinea pig plasma. This antibody also detected Factor VIII antigen in a solubilized protein mixture prepared from isolated cultured guinea pig megakaryocytes. Cultured guinea pig megakaryocytes were labeled with radio-active leucine. By radioautography, 96.2% of the radio-activity was present in megakaryocytes. The radio-active Factor VIII antigen present in the solubilized cell protein mixture was isolated by immunoprecipitation and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results demonstrate that cultured guinea pig megakaryocytes synthesize Factor VIII antigen which contains the same polypeptide subunit (mol wt 200,000) present in guinea pig plasma Factor VIII antigen.


Demonstration of lipids in the pathologic granules in cementum and dentin in periodontal disease. Granules can be demonstrated in dental cementum which has been exposed to the oral environment as a result of gingival retraction. In the present study frozen sections of decalcified, periodontally diseased teeth were stained by the Sudan black and Schultz-Hershberger methods, which indicated that the granules contained cholesterol or steroids. The lipid content of cementum suggests that the outer layer of cementum has been penetrated by exogeneous substances of bacterial or salivary origin.


A high molecular weight allergenic fraction of honeybee venom. Chromatography of honeybee venom on Sephadex G-150 super fine revealed a high molecular weight (HMW) fraction that elutes prior to hyaluronidase (HYAL) and comprises 2% to 4% of the venom weight. HMW appears to exist in polymeric form, and the polymer which is present in greatest concentration has an estimated molecular weight of 105,000 D. The 12% nitrogen content of HMW suggests it may not be all protein. HMW is antigenically and enzymatically distinct from HYAL and phospholipase A2 (PHOS A). The acid phosphatase activity known to be present in honeybee venom was found in the HMW fraction. Since it reacts by RAST with the sera of most individuals known to be sensitive to honeybee venom, and releases histamine from the peripheral leukocytes of such individuals, its role as an allergen is confirmed. Since individuals react to different degrees to HMW, HYAL, and PHOS A, there does not appear to be a single principal allergen in honeybee venom.


Human lung tissue and anaphylaxis. I. The role of cyclic GMP as a modulator of the immunologically induced secretory process. A close relationship between increased concentrations of cyclic GMP in human lung tissue and the capacity for acetylcholine to enhance the immunologic secretion of histamine and SRS-A has been found. Acetylcholine (10(-7) to 10(-11) M) produced parallel increases in both cyclic GMP and the immunologic release of mediators; the muscarinic blocking agent atropine prevented both responses. The increase in cyclic GMP in human lung after acetylcholine stimulation was apparent within 30 sec, peaked by 120 sec, and abruptly returned to control levels thereafter. The ability of acetylcholine to enhance the antigen-stimulated secretion of mediators followed the same time-course. PGF2alpha (3.3 X 10(-4) M to 3.3 X 10(-7) M) increased the cyclic GMP content of human lung tissue in a dose-related fashion. Pretreatment of IgE-sensitized lung tissue with acetylsalicylic acid (10 microgram/ml) had no effect on baseline cyclic nucleotide levels, the capacity for antigen to induce mediator release, or the increase in cyclic GMP and facilitation of the immunologic release of mediators produced by acetylcholine.


Monodispersal and deoxyribonucleic acid analysis of prostatic cell nuclei. We collected prostatic glands from 50 unselected autopsies at the Pathology Institute and compared their histologic sections with cytologic preparations and with results of photometric measurements of isolated prostatic cells and isolated nuclei. The results obtained with single cell photometry and flow-through cytophotometry proved to be comparable with one another and with the results of the cytologic and histologic studies. With these methods of cytophotometry we could differentiate inflammatory conditions, microcarcinomas and frank carcinomas from normal and hyperplastic prostatic tissue. We had difficulties, however, preparing adequate suspensions of cell nuclei from chronic fibrosing prostatitis. Our results indicate that it should be possible for diagnostic purposes to combine the technique of fine needle biopsy of the prostate with that of flow-through cytophotometry and to use the combined techniques for studying diseases of other organs.


Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples. Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.


Peculiarities of cytochemical properties of cancer cells as revealed by study of deoxyribonucleoprotein susceptibility to Feulgen hydrolysis. Chromatin of human squamous carcinoma cells reacts more intensively to short (1-2 min) acid hydrolysis in the Feulgen reaction and is, after such treatment, more intensively stained by Schiff reagent than chromatin of normal cells of the same origin. To reveal this difference in chromatin properties the use of a fluorescence variant of the Feulgen reaction is necessary because nuclei-binding of Schiff reagent after such short hydrolysis is so weak that the amount of the stain bound by means of absorption technique is hardly possible. The use of increased sensitivity of cancer cells chromatin to acid hydrolysis for cancer cytodiagnosis is suggested, especially for the diagnosis of so called diploid cancers for which detection on the basis of deoxyribonucleic acid content determination is impossible.


Analysis and sorting of living cells according to deoxyribonucleic acid content. The methods for measuring the deoxyribonucleic acid content of individual mammalian cells and sorting them on the basis of this parameter have until now required fixation or other treatment which renders the cells nonviable. Using a class of bis-benzimidazole dyes, Hoechst 33258 and 33342 and a multiparameter computer-controlled cell sorter, we have been able to stain and separate living cells in the G1, S, and G2+M phases of the cell cycle and to continue their growth in tissue culture with high retention of viability (greater than 90%) and no increase in heteroploidy. The quenching of the fluorescence of the bound dye by 5-bromodeoxyuridine incorporated into cellular deoxyribonucleic acid is being used with the flow system to detect and isolate mutants in deoxyribonucleic acid metabolism spectroscopically.


Computer-assisted monocyte esterase assay by flow-cytophotometry. A Wang model 2200 computer has been interfaced with the Bio/Physics Systems, Inc. model 6300 Cytograf and model 2100 Distribution Analyzer. Using a custom designed software program, in conjunction with an azo-dye technic for staining monocytes for nonspecific esterase activity, it has been possible to obtain rapid and reliable data concerning relative values for intracellular monocyte esterase activity. The method is based on measuring the axial light-loss voltage signal for each of one thousand stained monocytes. Individual stained monocytes were assigned to one of four groups (A, B, C, D), dependent upon the magnitude of the signal and were given different rating values (1, 2, 3, 4) according to their group designation. A "score" was derived for each blood sample by multiplying the percentage of cells (monocytes) in each group category by the appropriate factor and summing these values. The technic permits rapid objective assessment of intracellular nonspecific esterase activity in monocytes suspended in a mixed cell population. Both Gaussian and bi-modal patterns for monocyte esterase were observed. The latter suggests a dual monocyte population.


Virometer: an optical instrument for visual observation, measurement and classification of free viruses. An instrument based on fluorescence correlation spectrometry and total reflection fluorescence visually and photoelectrically detects and sizes viruses at moderate concentrations in biologic fluids in minutes. Viruses can be classified using their nucleic acid type and amount determined by new fluorescent staining and data handling techniques.


Multiparameter geometric and densitometric analysis of the G0-G1 transition of WI-38 cells. Automated image analyses were performed using Feulgen stained smears of WI-38 cells that were either confluent, or that had received a nutritional stimulus to proliferate 3 hr before collection. These experiments show that it is possible to observe changes in morphometric and densitometric parameters of nuclei that correlate with structural and functional differences in isolated chromatins from quiescent G0 and proliferation G1 cells that have been demonstrated by other means. Scatter plot analyses of the data indicated the presence of nuclear images from the stimulated G1 population that had the same deoxyribonucleic acid content as the confluent G0 cells, but had greater areas, perimeters and horizontal projections and smaller mean free paths, form factors, and average optical densities. Multiparameter cluster analysis permits, even minimally, an objective, model-independent identification of G0 from G1 cells that present an increased nuclear dispersion (i.e., lower average optical density) systematically accompanied by increased nuclear convolution (i.e., lower form factor), both compatible with the reported increase in available binding sites with respect to G0 cells.


Automatic measurement of sister chromatid exchange frequency. An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.


Measurement of mammalian sperm deoxyribonucleic acid by flow cytometry. Problems and approaches. Measurement of mammalian sperm deoxyribonucleic acid content is of importance in several areas of biomedical research. When measured in flow systems with orthogonal axes of illumination, flow and detection, an unexpected, distorted distribution consisting of a narrow peak with a lateral extension to the right is observed. Several lines of evidence lead to the conclusion that this effect is an optical-geometric artifact attributable to the flat shape and high index of refraction of mammalian sperm heads. This artifact disappears when an epiillumination flow system is used in which the optic axes for illumination and detection and the flow axis are all coincident. Other approaches also eliminate the artifact. The resulting coefficients of variation observed after acriflavine-Feulgen staining are 4-5%, short of the goal of 1.5% required to distinguish between human sperm bearing X and Y chromosomes and to develop a mutagen test system using mice.


Flow system fluorescence polarization measurements on fluorescein diacetate-stained EL4 cells. We have adapted a multiparameter cell sorter to measure the distribution of fluorescence polarization in cell populations. Measurements carried out on EL4 cells show that the percent polarization of fluorescein fluorescence decreases with increasing fluorescence intensity. This inverse relationship between polarization and intensity is shown both within the cell population and by the average values of the two quantities during both the increase and decrease of fluorescence intensity. The quantitative relation between intensity and polarization is different in hypertonic than in isotonic media. These results suggest that polarization measurements carried out at a fixed time after incubation of cells with fluorescein diacetate, which is converted to fluorescein within the cells, may depend in part on the rate of fluorescein accumulation, and that agents that have been reported to change the polarization of fluorescein in living cells may do so by changing the kinetics of fluorescein accumulation.


Recognition of cells in mitosis by flow cytofluormetry. Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.


Differential fluorochromasia of human lymphocytes as measured by flow cytometry. Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes.


Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry. Fluorometric detection of the biosynthetic incorporation of 5-bromodeoxyuridine (BrdU) into deoxyribonucleic acid has permitted cytologic studies of chromosome structure, replication, and repair. Some of these phenomena, previously detected using BrdU-dye techniques on fixed microscopic preparations, should be particularly amenable to analogous experimentation in fluorescence flow systems. Problems involved in interfacing BrdU-dye methodology with flow fluorometry are discussed. The effects of certain chemical modifications of bisbenzimidazole dyes on their spectroscopic properties and potential use for detecting BrdU incorporation into unfixed cells are described. Data on the use and energy transfer characteristics of a pair of deoxyribonucleic acid-binding dyes (33258 Hoechst and ethidium bromide) capable of simultaneously providing information about BrdU substitution and total deoxyribonucleic acid content are presented.


Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst. This paper describes a flow-cytometric application of the quenching of fluorescence from 33258 Hoechst stained Chinese hamster ovary-line cells due to the incorporation of 5-bromo-deoxyuridine (BrdU) into the cellular deoxyribonucleic acid. Cells were grown for 24 hr in medium containing BrdU in concentrations ranging from 1 x 10(-8) to 1 x 10(-4) M. For each concentration we measured the average fluorescence as determined by flow cytometry, the  extent of BrdU substitution and the effect of the BrdU on cell growth. We determined that a BrdU concentration of 1 x 10(-5) M resulted in sufficient substitution to quench the fluorescence from 33258 Hoechst by a factor of 4, allowing discrimination between cycling and noncycling cells. The extent of BrdU substitution after growth for 24 hr in this concentration of BrdU was 64%. These data indicate the feasibility of detecting deoxyribonucleic acid synthesis in whole cells using the 33258 Hoechst-BrdU methodology.


The automation of cytochemical methods for automated cytophotometers. The development of an automated differential white blood cell counter is reviewed. After the red cells have been lysed, the white cells are counted by staining and passing through an electro-optical chamber in liquid suspension, surrounded by a laminar, or sheath, stream. Staining procedures were made specific for each type of leukocyte, and separate channels were used for counting each type. Staining intensity and characteristics of the various types of blood cells are discussed. They relate to enzyme levels and the effect on differentiation and identification of the cells. Since reasons for some of the design features are not obvious, discussion of the relevant problems is included. Several applications that go beyond routine differential counting are described.


Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes. Requirements for flow cytometry of metaphase chromosomes stained with three deoxyribonucleic acid (DNA)-specific fluorescent dyes--Hoechst 33258, Chromomycin A3, and ethidium bromide--are reviewed. Fluorescence properties of these three stains when bound to mitotic cells or to chromosomes in suspension are measured and compared with fluorescence properties when bound to DNA in solution. Conditions are given for high resolution flow cytometry of Chinese hamster chromosomes stained with each of the fluorophors, and histograms are presented that exhibit differences in relative peak position and area. Energy transfer fluorescence between two DNA stains is presented as a potentially useful new parameter for flow cytometry of chromosomes and is illustrated by fluorescence energy transfer from Chromomycin A3 to ethidium bromide when simultaneously bound to hamster mitotic cells.


The application of cytochemistry to automation. The application of cytochemical techniques for use with automated instruments introduces a variety of problems not encountered with traditional cytochemical stains prepared for human visualization. This is particularly true for flow-cytophotometers, which require that cells be stained in suspension, making it difficult to remove reagents once they are added to a staining mixture. It is much less a problem for pattern recognition instruments. The major difficulties relate to cell preparation, fixation, staining, controls and interpretation of instrumental data. The new cytochemical techniques or modifications of existing methods that are required for use with such instruments present a fresh challenge to cytochemists. The need for such methods is great and the rewards may be equivalent.


Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T cell-dependent proliferative response with lymph node cells from primed mice. An in vitro assay which measures antigen-induced proliferation of primed murine lymph node cells is described. The response is mediated by T eclls since it can be obtained by using nylon wool-passed lymphocytes (less than 1% Ig+ cells) and it can be abolished by treatment with anti-Thy 1.2 and C. Furthermore, LN cells from nu/nu mice injected with antigen do not demonstrate antigen-induced proliferation in contrast to the response observed in euthymic littermate controls. Other relevant parameters of this proliferative assay include the observations that the response is highly antigen specific, can be seen as early as 4 days and as late as 60 days after in vivo priming, is restricted to the use of certain sets of LN when animals are injected subcutaneously at the base of the tail, and can be seen with LN cells from mice primed with antigen in either CFA or ICFA. The ease of the assay coupled with its specificity and quantitative dimensions provides a direct and simple method to evaluate processes involved in antigen-induced murine T lymphocyte activation.


Antibody-dependent cellular cytotoxicity against murine leukemia viral antigens: studies with human lymphoblastoid cell lines and human peripheral lymphocytes as effector cells comparing rabbit, goat, and mouse antisera. A thymic lymphoblastoid cell line derived from a New Zealand Black mouse produces murine leukemia virus (MuLV) and was used as a target in model systems for the in vitro study of antibody-dependent cellular cytotoxicity (ADCC). Several human lymphoblastoid cell lines were investigated as potential effector cells. The most promising (Raji cells) bound to antibody-coated target cells but caused only modest levels of ADCC at 25:1 effector-to-target cell ratio with substantial lysis in the absence of antiserum. Human peripheral lymphocytes were active as effector cells in ADCC at a 5:1 ratio and produced no lysis in the absence of antibody. These cells were used to demonstrate that high dilutions of rabbit antisera to MuLV antigens p30, p15, p12, and p10 were capable of mediating lysis of MuLV-producing target cells but not of a virus-negative murine cell line. A murine antiserum to Thy 1.2 and three caprine antisera to MuLV antigens that were active in complement-mediated cytotoxicity functioned poorly in inducing ADCC; however, rabbit antisera to similar antigens were 16- to 512-fold more efficient in cell-mediated than in complement lysis. The inefficiency of goat antisera was not due to shedding of cell surface antigens or generation of blocking factors but rather to lack of lytic interaction of antibody-coated targets with the effector cells.


The frequency of para-azophenylarsonate and dimethylaminonapthalene-sulfonyl-specific B cells in neonatal and adult BALB/c mice. The limiting cell dilution splenic focus technique has been used to determine the frequency of p-azophenylarsonate (Ars) and 5-dimethylaminonaphthalene-sulfonyl (Dansyl)-specific B cells in neonatal and adult mice. The adult frequencies for Ars and Dansyl in conventionally reared BALB/c mice are 1.5 +/- 0.3/10(5) B cells and 9.4 +/- 3.3/10(5) Bcells, respectively. Dansyl-specific precursor cells are present in high frequencies at birth and reach adult precursor frequency levels during the first week of life. This finding mirrors the ontogenetic development previously described fro dinitrophenyl (DNP) and trinitrophenyl (TNP), but contrasts with the results obtained fro fluorescein (FL) and phosphorylcholine (PC), for which precursors arise later in development. On the other hand, the kinetics of appearance of B cells specific for Ars is unique in that the precursor frequency appears to decrease during the first week of life. The data presented remain consistent with a highly ordered, rigorously predetermined acquisition of the specificity repertoire during ontogeny. By increasing the flexibility of the splenic focus technique to allow analysis of other, chemically diverse haptens, results have been obtained that confirm and extend observations made with DNP, TNP, FL, and PC.


Immune responses of inbred guinea pigs to the sequential polymer poly(L-Tyr-L-Ala-Gly): studies with the oligomers of the polymers. Synthetic known sequence polypeptides poly(Tyrosine-Glutamic acid-Alanine-Glycine) T-G-A-Gly), were found to be very immunogenic in responder inbred guinea pigs. Two and one-half micrograms were enough to elicit both humoral and cellular responses. Only the alpha-helical oligomers were immunogenic and were able to inhibit the homologous antigen-antibody reactions. The random polymers of comparable amino acid composition, i.e., poly(glutamic acid60alanine40) (GA), poly(glutamic acid50 tyrosin50) (GT), poly(glutamic acid60alanine30tyrosine10)(GAT10), did not inhibit. The antibodies against (T-G-A-Gly)n did not bind to the closely related known sequence polymer poly tyrosine-alanine-glutamic acid-glycine) (T-A-G-Gly)n or to the above random polymers. It is thus concluded that antibodies against (T-G-A-Gly)n are directed against conformational determinats.


The formation of active hybrid immunoglobulins from the heavy and light chains of beta(1, 6) D-galactan binding murine myeloma IgA's S10 and J539. Murine myeloma immunoglobulin (IgA, K) J539, which shows enhanced tryptophanyl fluorescence on ligand binding, and S10, which shows reverse-sign changes in tryptophanyl fluorescence on ligand binding (RLIF, see below), have been reduced, alkylated, and dissociated into their light (L) and heavy (H) chains. Two hybrid recombinants, H10L539 and H539L10, have been prepared and the 7S material has been isolated by chromatography. The binding behavior of these recombinants was studied with a number of ligands. Both recombinants showed activity with beta(1 leads to 6) linked galactose ligands comparable to the native immunoglobulins. The ligand-induced fluorescence changes of the recombinants paralleled those of the heavy chain donor. For the recombinant H10L539, two different galactose-ligands caused fluorescence changes in opposite directions. It was quantitatively shown that binding of these ligands, nevertheless, took place in the same combining region. The idiotype of each recombinant resembled that of the heavy chain donor.


Reduced severity of lesions in mink infected transplacentally with Aleutian disease virus. Inoculation of mink late in the second trimester of pregnancy with Aleutian disease virus (ADV) produces a persistent infection in the offspring. When these mink were analyzed at 83 days of age and compared with adolescent mink infected for a similar length of time, the transplacentally infected mink show: 1) a marked reduction in plasmacytosis, immunoglobulin level and specific ADV antibody; 2) increased amounts of infectious ADV and numbers of cells containing viral antigen; 3) a marked reduction in immune complex glomerulonephritis and absence of immune complex arteritis; 4) free ADV antigen in the glomeruli; and 5) a striking accumulation of eosinophils in the tissues. The findings suggest that the degree of ADV expression is partially immunologically controlled.


Effects of C-reactive protein on platelet function. III. The role of cAMP, contractile elements, and prostaglandin metabolism in CRP-induced inhibition of platelet aggregation and secretion. It was previously demonstrated that C-reactive protein (CRP) inhibits platelet aggregation and release reactions, activation of platelet factor 3, and platelet-dependent clot retraction. Multiple considerations including selective inhibition of secondary wave aggregation suggested that CRP exerted its inhibitory effects by interfering with the release of endogenous ADP. In the present investigation, CRP was found by direct assay to inhibit the release of endogenous ADP and/or serotonin concomitant with inhibition of platelet aggregation stimulated by ADP, epinephrine, thrombin, and AHGG. CRP did not induce an increase in the basal level of platelet cAMP, suggesting independence of a direct effect upon this mediator system. Furthermore, CRP did not inhibit the aggregation and secretion induced by the antibiotic ionophore A23187, suggesting the absence of a direct effect upon the activation of platelet contractile elements. By contrast, CRP did inhibit both thrombin-induced release of malondialdehyde, a prostaglandin endoperoxide nonprostanoate endproduct, and platelet aggregation induced by the prostaglandin endoperoxide precursor arachidonic acid. These data, therefore, raise the possibility that CRP inhibits platelet reactivities by interfering with an aspect of porstaglandin metabolism, and that this occurs subsequent to the hydrolytic accumulation of arachidonic acid and prior to the movement of calcium from the platelet dense tubules. These studies support the concept that CRP serves to modulate platelet reactivities during acute inflammatory reactions.


Development of a hemolytic plaque assay for glutamic acid, lysine-containing polypeptides: demonstration that nonresponder mice produce antibodies to these peptides when conjugated to an immunogenic carrier. Fowl gamma-globulin, when chemically conjugated to GLO or GL, functions as a T-dependent immunogenic carrier and stimulates anti-GLO and anti-GL antibody production in nonresponder mice. The conjugation procedure utilizes the Schiff base reaction. The anti-GL and anti-GLO responses were detected by hemagglutination and hemolytic plaque assays by using GL-coated erythrocytes. The coupling of GL to erythrocytes utilizes a novel procedure in which a palmitoyl derivative of GL is adsorbed onto red blood cells. The optimal conditions for preparing the palmitoyl derivative and for coupling to SRBC are presented. With the hemolytic plaque assay, we have verified that GLO responder animals make both IgM and IgG responses, whereas nonresponder mice fail to make either IgM or IgG plaque-forming cells.


The role of T cells in anti-herpes simplex virus immunity. I. Induction of antigen-specific cytotoxic T lymphocytes. Mice infected with herpes simplex virus develop little or no cytotoxic T lymphocyte (CTL) response. However, in lymph nodes (LN's) draining a local site infected with HSV, antigen-specific CTL precursors are sensitized, which upon transfer to in vitro culture conditions develop within 72 hr into effective CTL. The in vivo blockade of CTL differentiation can be overcome by cyclophosphamide, suggesting that a cyclophosphamide-sensitive mechanism blocks the in vivo generation of HSV-immune CTL. The cytolytic activity of HSV-immune CTL is H-2 restricted and antigen specific. Thus CTL sensitized toward HSV type 1 discriminate between syngeneic targets infected with either the immunologic HSV variant type 1 or type 2 (and vice versa). H-2-matched target cells exposed for 30 min to infectious HSV are lysed within 60 min of contact with CTL. Since HSV replication is believed to require more than 4 to 5 hr, the data suggest that either the expression of HSV-dependent "early proteins" takes place within 30 to 90 min or cell membrane-integrated HSV virion represents the target antigen of CTL.


In vitro sensitization of human lymphocytes against histiocytic lymphoma cell lines. I. Primary sensitization of lymphocyte subpopulations. Peripheral blood lymphocytes from normal donors expressed spontaneous cytotoxic activity against human diffuse histiocytic lymphoma cell lines. In the unfractionated state, they could not be further sensitized in vitro against these cell lines. By applying cell separation techniques before culture, subpopulations of lymphocytes were obtained which could be sensitized in vitro and manifested cytotoxic activity against human histiocytic lymphoma cells. Three methods of separation were found effective: E rosette enrichment; elimination of Fc receptor positive cells; and removal of nylon wool adherent cells. Under these conditions, cross-reactive cytotoxicity was observed against non-neoplastic lymphoblastoid cell lines, but not against normal lymphocytes.


Immune ascites in the guinea pig: specificity of cells and antibody in an induced peritoneal exudate. Strain 2 guinea pigs, immunized with Lys10-Lys(Dnp) in CFA and repeatedly injected intraperitoneally with adjuvant, developed ascites. The fluid was harvested over 8 months in total amounts up to 2 liters per animal and contained substantial amounts of cells and antibody which reacted with the immunizing antigen and related peptides. The antibody was of the IgG and IgA classes and showed restricted heterogeneity. Among synthetic Dnp-oligopeptides, both the cells, studied by antigen-stimulated thymidine incorporation, and the purified antibody, studied by fluorescence quenching, demonstrated the same specificity for the immunizing antigen as has previously been noted in lymph node cells and in serum antibody. The technique offers a means for studying more cells and more antibody than has previously been possible from individual guinea pigs.


Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype. The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.


Structural and genetic studies on chicken 7S immunoglobulin allotypes. III. Proposed nomenclature for the CS-1 gene alleles. A survey of 47 inbred or partially inbred chicken lines derived from five sources in the United States and Europe revealed considerable genetic polymorphism in the structural gene (CS-1) responsible for the production of the predominant chicken 7S Ig heavy chain. A minimum of 10 alleles of the CS-1 gene were detected as unique combinations or phenogroups of CS-1 specificities. A system of nomenclature for CS-1 alleles was developed and six homozygous lines were designated as prototype lines. The remaining four CS-1 alleles occurred only in lines that were polymorphic for 7S Ig allotypic specificities.


Immunosuppressive properties of a peptic fragment of BSA. The immunogenic properties of a peptic fragment of BSA were investigated. BSA was subjected to limited proteolysis by pepsin and the resulting fragments were separated on DEAE cellulose. The fragment under consideration, Fraction Ia (m.w. 8000 to 10,000), did not precipitate with anti-BSA serum but did inhibi, the binding of specific antibody to labeled BSA, indicating the presence of determinants found on the native antigen. BDF1 mice immunized with Fraction Ia in A1 (OH)3 gel or in complete Freund's adjuvant produced no significant antibody response as measured by passive cutaneous anaphylited a (PCA) or by a modified Farr assay. The fragment elicited a PCA reaction in mouse skin sensitized with anti-BSA serum. Treatment of mice with single doses of Fraction Ia at various time intervals before immunization with BSA resulted in significant suppression of the formation of anti-BSA antibody. The conditions of suppresion of the IgE response by the peptic fragment were studied in greater detail. Evidence is presented that such suppression can be attributed to the presence of specific T suppressor cells in our system.


Immunoregulation of experimental allergic encephalomyelitis: conditions for induction of suppressor cells and analysis of mechanism. We determined requirements for the induction of immunoregulatory suppressor cells in experimental allergic encephalomyelitis (EAE) in Lewis rats. Pretreatment of rats with myelin basic protein (BP) in incomplete Freund's adjuvant (IFA) stimulates the proliferation of suppressor cells that localize in lymph nodes and spleen (but not thymus) and exert control over the development of clinical EAE. Dosage studies revealed that 3 X 10(7) suppressor cells can adoptively transfer suppression to syngeneic recipients. Transferred unresponsiveness wanes within 3 weeks, indicating that the suppressor cells are short-lived lymphocytes, although actively induced unresponsiveness persists for at least 8 weeks, probably as a result of continual proliferation under the influence of antigen. No evidence was obtained to suggest that antigen carry-over or blocking antibody production accounts for adoptive transfer of unresponsiveness. Suppressor cells apparently act at the inductive phase of the immune response since they had no inhibitory effect on adoptive transfer of disease by effector lymph node cells. Other mechanisms also may play a role in unresponsiveness to EAE, since rats pretreated i.v. with high dosages of soluble BP were temporarily rendered unresponsive, although suppressor cells could not be detected in these animals.


The demonstration of subtype (D or Y)-specific determinants on the surface of the presumed hepatitis B virus. Dane particles isolated from the sera of HBsAg/ad and HBsAg/ay carriers were reacted with monospecific antibodies to the d and y subtype-specific determinants of HBsAg/. Dane particles from HBsAg/ad expressed the d determinant on their surfaces and those from HBsAg/ay sera contained the y specificity. Both complete (DNA-P and HBcAg) and defective (HBcAg alone) Dane particles expressed the subtype-specific determinants.


Studies on non-H-2 linked lymphocyte activating determinants. I. Description of the cell type bearing the Mls product. The non-H-2 linked, lymphocyte activating determinants (LADs), which are coded by the minor (histocompatibility) lymphocyte stimulating locus (Mls), were detected on B lymphocytes, but not on immunoglobuin (Ig) negative or on theta positive T lymphocytes. These determinants, like the complement receptor and surface IgD, develop late in neonatal life. Thus, the Mls coded LADs mark a population of B lymphocytes which are not present in the spleens of newborn to 2-week-old mice. Studies of B lymphocytes derived from the spleens of adult mice indicated that the cells bear the Mls coded LADs have a high density of surface Ig and possess the complement receptor.


A polyvalent human gamma-globulin immune to Pseudomonas aeruginosa: passive protection of mice against lethal infection. As a means to development of guidelines for therapeutic application to human disease, preparations of human polyvalent gamma-globulin immune to Pseudomonas aeruginosa (PG) were studied in acute infections in mice. PG was highly effective in controlling lethal infections induced in mice by the major immunotypes of P. aeruginosa; greater than or equal to 10 microgram of of gamma-globulin per mouse protected against challenge with less than or equal to 10(6) 50% lethal doses of P. aeruginosa. PG was less than or equal to 57 times more effective than normal human gamma-globulin. The active antibody component is specific for each immunotype; it is of the IgG type and undoubltedly is directed against the O-antigen. PG was was not protective against challenge with Escherichia coli, Enterobacter cloacae, Proteus mirabilis, or Klebsiella pneumoniae; a low degree of cross-protection was seen against Serratia marcescens. In a model infection involving mice in a terminal stage of advanced P. aeruginosa infection, human plasma immune to P. aeruginosa proved ineffective, but the gamma-globulin component showed moderate activity. The apparent irreversibility of this late-stage infection is not clearly ascribable to a toxin. It is postulated that the successful treatment of advanced P. aeruginosa infections in humans would require multiple therapeutic approaches, including passive immunization with a high-potency, specifically immune globulin.


Immunological investigation of infants with septicemia or meningitis due to group B Streptococcus. Purified polysaccharide from type III group B Streptococcus contains both a type III-specific determinant and another determinant that is common to strains of serotypes other than type III. The polysaccharide contains sialic acid, galactose, heptose, glucose, glucosamine, and mannose. Serum antibody to this antigen was measured by means of a radioactive antigen-binding assay. Sera from 36 (67.9%) of 53 women with healthy newoborns contained antibody, a prevalence significantly different from that in sera from 15 women (13.3%) whose neonates developed septicemia or meningitis due to type III group B Streptococcus. Complete concordance for presence or absence of anticapsular antibody in sera from 14 women at delivery and in their neonates' cord sera was demonstrated; this concordance indicates transplacental transfer of antibody. Sera from each of four adults with invasive infection who were studied during convalescence contained antibody to the capsular polysaccharide of type III group B Streptococcus. In contrast, antibody was absent from 10 infants who had recovered from bacteremia, septicemia, and/or meningitis due to type III group B Streptococcus.


Antibody to influenza virus matrix protein detects a common antigen on the surface of cells infected with type A influenza viruses. Antisera to the type-specific internal influenza virus matrix (M) protein of a type A influenza virus were produced in goats. In the presence of complement, anti-M serum was cytotoxic for target cells which were infected with a variety of serologically distinct type A influenza viruses, but did not react with type B influenza virus-infected cells. Absorption experiments indicated that anti-M serum detected a common antigen(s) on the surface of type A-infected cells. This serological cross-reactivity parallels the cross-reactivity observed for the cytotoxic T-cell response to type A viruses.


Antigen presentation in the murine T-lymphocyte proliferative response. I. Requirement for genetic identity at the major histocompatibility complex. A method is described for stimulating proliferation in primed populations of murine T lymphocytes using antigen bound to mitomycin-C-treated spleen cells. This form of antigen presentation appears to be an active process because heat-killed spleen cells are ineffective, and because genetic similarity at the major histocompatibility complex (MHC) between the responder T cells and the presenting spleen cells is required for effective interactions. At all times examined, from day 3 to day 6 of the proliferative response, syngeneic spleen cells presented antigen better to peritoneal exudate T-lymphocyte-enriched cells (PETLES) than semisyngeneic F(1) spleen cells, which in turn could present antigen better than totally allogeneic spleen cells. Spleen cell mixing experiments demonstrated that these genetic restrictions were not the result of suppression by the ongoing mixed lymphocyte reactions (MLR) in the allogeneic and F(1) cases. Furthermore, incompatibility at the Mls locus generated a strong MLR but failed to prevent antigen presentation if the spleen cells and PETLES were compatible. Genetic mapping studies demonstrated that compatibility at only the I-A subregion of the MHC was sufficient for effective presentation of the antigen, dinitrophenylated ovalbumin. Compatibility at only the K region, or the K and D regions was not sufficient. These results support the concept that functional activation of primed, proliferating T lymphocytes requires the participation of gene products coded for by the I region of the MHC. This conclusion is consistent with a growing body of evidence which suggests that most T cells recognize antigen in association with MHC gene products.


Cytotoxic T-cell response to Ectromelia virus-infected cells. Different H-2 requirements for triggering precursor T-cell induction or lysis by effector T cells defined by the BALB/c-H-2db mutation. The T(c)-cell response to ectromelia virus infection was studied in BALB/c-H-2(db) mice which carry a loss mutation in the H-2D region that results in the absence from cell surfaces of a molecule (D') bearing certain public H-2 specificities. When infected, these mice showed a poor response of T(c) cells that recognize H-2D(d) plus virus-specific determinants on infected macrophage targets, but gave a normal response to H-2K d plus virus-specific antigens. However, their own infected macrophages do display wild-type antigenic patterns involving virus and H-2D(d) since they were killed as efficiently as wild-type (BALB/c,H- 2(d))-infected cells by T(c) cells specific only for H-2D(d) plus viral antigens. When tested in vitro, infected BALB/c-H-2(db) cells stimulated a poor T(c)-cell response to H-2D plus virus-specific antigens, but stimulated a normal response (in comparison with infected BALB/c macrophages) to H-2K(d) plus viral antigens. Uninfected BALB/c-H-2(db) cells stimulated a normal T(c)-cell response to minor H antigens or trinitrophenyl in association with H-2D(d), thus suggesting that the defective response to infection may reside in a failure of the relevant H-2D(d) antigens of mutant cells to physically associate with viral antigens. Close association of viral and H-2D-coded molecules was also suggested by ability of specific anti-H-2K or -H-2D to partially block T(c)-cell-mediated lysis of infected targets. These results were interpreted to mean that H-2Dd-dependent, virus- immune T(c) cells recognized an antigenic pattern consisting of virus- specific and H-2D(d) determinants with the latter borne on an H-2D molecule carrying serologically-defined H-2D(d) private specificities. A second H-2D(d)-coded molecule (D') was not required for recognition and lysis by activated T(c) cells, but was apparently necessary for efficient stimulation of precursor T(c) cells, perhaps by promoting appropriate physical association of viral and H-2D(d) molecules.


Plasmin inhibitor interactions. The effectiveness of alpha2-plasmin inhibitor in the presence of alpha2-macroglobulin. alpha2-Plasmin inhibitor and alpha2-macroglobulin were allowed to compete for the protease plasmin. The binding of the enzyme to these inhibitors was assessed by two different but comparable methods. The interactions were completed in 10 s of incubation, and transfer of plasmin from one inhibitor to the other did not occur. Almost as much plasmin was bound to alpha2-plasmin inhibitor in mixtures containing a large molar excess of alpha2-macroglobulin relative to plasmin or alpha2-plasmin inhibitor, as was bound in mixtures not containing alpha2-macroblobulin. These studies demonstrate directly the effectiveness of alpha2-plasmin inhibitor in binding and inhibiting plasmin in the presence of alpha2-macroglobulin, and suggest that the alpha2-plasmin inhibitor may be the major circulating plasmin inhibitor.


Serological distinciton of heavy chain variable regions (VH subgroups) of mouse immunoglobulins. I. Common VH determinants on the heavy chains of mouse myeloma proteins having different binding sites. 16 of more than 100 mouse myeloma proteins, including 3 proteins of the IgG2a class and 13 of the IgA class, were shown to have a similar heavy chain variable region (VH) antigen(s) (U10-173). The proteins bearing these antigenic determinants (U10-173+ proteins) represented at least five different ligand-binding specificities. These findings, taken togeter with available sequence data for VH regions of U10-173+ proteins, have led us to conclude that U10-173 defines a small number of related VH subgroups. The ability to detect VH subgroups in mice by serological means, as has been done in humans also, promises new and useful kinds of VH markers for immunologic study.


Specificity of cytotoxic effector cells directed against trinitrobenzene sulfonate-modified syngeneic cells. Failure to recognize cell surface-bound trinitrophenyl dextran. Mouse splenic lymphocytes and lymphoid tumor cells were modified with the trinitrophenyl (TNP) group either by treatment with trinitrobenzene sulfonate (TNBS) (which covalently modifies cell surface proteins) or with TNP stearoyl dextran (TSD) (which binds to the cell by noncovalent forces). These cell preparations were compared for their ability to: (a) sensitive syngeneic splenic lymphocytes leading to the generation of cytotoxic effector cells; (b) serve as lysable targets in a 4-h(51)Cr- release assay for effector cells generated in (a); and (c) act as blocking cells in the lysis of TNBS-medified targets lysed by TNP self effector cells generated in (a). In none of these three experimental systems did TSD-medified syngeneic spleen or H-2-matched tumor cells act either as a sensitizing immunogen or as a target antigen, despite the demonstration that quantitatively equivalent mounts of TNP were exposed on the cell surface in the TNBS- and TSD-modified cells. In contrast, TNBS-modified spleen cells sensitized syngeneic lymphocytes to generate effectors against TNBS-modified syageneic targets. Furthermore, TNBS- modified, H-2-matched cells served as specific lysable targets and as inhibiting cells for such effectors. These results indicate that the manner in which TNP is associated with the cell surface is important in the immunogenicity and antigenicity of hapten-modified syngeneic stimulating cells in generating H-2-associated cell-mediated lympholysis (CML) reactions. These findings raise the possibility that a covalent or at least a stable linkage with cell surface proteins (possibly H-2- controlled products) is important for immunological function. Furthermore, these observations do not favor the dual receptor model for H-2-restricted syngeneic CML if it is assumed in such a model that one receptor is specific for the TNP moiety and the second for unmodified self major histocompatibility products.


Streptococcal M protein extracted by nonionic detergent. II. Analysis of the antibody response to the multiple antigenic determinants of the M-protein molecule. Purified streptococcal M protein extracted by nonionic detergent was used in an RIA and a solid-phase radiocompetitive inhibition assay to determine the nature of the immune response in both human beings and hyperimmunized rabbits to this complex antiphagocytic antigen. Results indicate that a type-specific response to an M antigen with the development of opsonic antibodies is the result of antibodies directed against the majority of the antigenic determinants of the molecule. Cross-reactions between certain M types on the other hand, are represented by antibodies directed against only a small percentage of these antigenic determinants. Results also suggest that avidity may play a role in the action of opsonic antibodies. However, the data indicate that factors besides avidity (i.e. sites bound by the antibodies) also seem essential for opsonization.


Active and passive cation transport and L antigen heterogeneity in low potassium sheep red cells: evidence against the concept of leak-pump interconversion. Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.


Purification and characterization of precipitating antigens from Theileria parva. Precipitating antigens from Theileria parva have been partially purified. Two antigens from each of the schizont and piroplasm stages of the parasite were identified; the major antigens from the two stages shared the same specificity. The antigens showed considerable molecular heterogeneity, almost certainly a result of the preparative method, and they always contained large amounts of DNA. The piroplasm antigens were of parasite nuclear origin and the schizont antigens were probably of the same origin. The antigens were weakly antigenic, and the activity against them of humoral antibody from cattle immune to East Coast fever was low. These antigens do not appear to induce protection against East Coast fever.


A comparative study of the 'rhodochrous' complex and related taxa by delayed-type skin reactions on guinea pigs and by polyacrylamide gel electrophoresis. Cell extracts prepared by ultrasonic disruption of 17 strains of the 'rhodochrous' complex and related taxa were compared by polyacrylamide gel electrophoresis and for immunologic relatedness, by skin test reactions. Two organisms, Jensenia canicruria and Nocardia calcarea, gave similar gel patterns and skin test reactions, and are considered to be identical. Extracts of nocardia rubra showed a strong antigenic relationship with those of three Nocardia pellegrino organisms (N325, N324 and N420) previously assigned to the 'rhodochrous' complex. Two Gordona organisms appeared to be less antigenically related to the 'rhodochrous' complex. Extracts of three of four organisms designated Lspi (Rhodococcus coprophilus Rowbotham & Cross 1976) elicited skin test reactions similar to those of the 'rhodochrous' strains. One Lspi strain, N650, showed striking similarities to the 'rhodochrous' complex strain N420 (Nocardia pellegrino).


Production of antiserum to the reverse transcriptase of Mason-Pfizer monkey virus. The reverse transcriptase of Mason-Pfizer monkey virus (M-PMV) has been isolated and partially purified by ion exchange chromatography. Sera from rabbits immunized with the partially purified enzyme have been shown by microimmunodiffusion analysis to be immunologically specific for the M-PMV polymerase. The immune serum also specifically inhibits M-PMV polymerase activity and this inhibitory activity has been shown to reside in the IgG fraction of the serum. The application of these reagents to examining virus identity and investigating the possible viral aetiology of human breast cancer is discussed.


Observations on the antigenic relationships between Epstein-Barr virus and herpesvirus saimiri. The antigenic relationships between Epstein-Barr (EB) virus and herpesvirus saimiri (HVS) have been investigated in comparative immunofluorescence, microimmunodiffusion and serum neutralization tests. No similarity was detected between the structural antigens of the two herpesviruses but the results of microimmunodiffusion tests showed that they shared a non-structural, virus-determined antigen. The implications of this finding are discussed in relation to the importance of HVS and its monkey lymphoma as a model system of herpesvirus oncogenesis.


Activation of an endogenous C-type RNA virus in rat embryo cells after transformation by herpes simplex virus types 1 and 2. Reverse transcriptase activity was detected in the supernatants of rat embryo fibroblast cell cultures transformed by HSV types 1 and 2 at either the sub-optimal temperature of 20 degrees C or the supra-optimal temperature of 42 degrees C. Rat cells clones which had been transformed at 20 degrees C contained higher levels of C-type virus DNA polymerase than did cell clones which had been transformed at 42 degrees C. Syncytia formation typical for C-type RNA viruses occurred at passages higher than 24. The activation of endogenous C-type RNA viruses was independent of the virus and transformation method used.


Similar polypeptide composition of fast-transported proteins in rat motor and sensory axons. SDS-polyacrylamide gel electrophoresis was used to characterize labeled proteins transported in rat motor and sensory axons after application of 3H-leucine to the neuron cell bodies. Two types of experiments were performed: first, transported protein accumulating proximal to a ligature placed on the sciatic nerve was analyzed; second, the segment of sciatic nerve nearest to the "wavecrest" of transported protein travelling down the nerve was analyzed. In both cases, no significant differences in peak position or amplitude were found in gels containing labeled proteins from motor or sensory axons. This may mean that the majority of fast-transported protein is involved in an axonal function common to the two types of neuron.


Characterization of proteins transported at different rates by axoplasmic flow in the dorsal root afferents of rats. Proteins synthesized by soma located in L4 dorsal root ganglia and supplied to the axonal branches extending centrally in the dorsal root and peripherally towards the sciatic nerve were analyzed for radioactivity following injections of [3H] leucine into the L4 dorsal root ganglia. All proteins located in the dorsal root and sciatic nerve were analyzed by SDS acrylamide gel electrophoresis at various times post injection. The differences in radioactivity between the dorsal root and sciatic nerve proteins were mainly quantitative and not qualitative, with many proteins of various molecular weight ranges being transported into both segments. Generally, it appears that in both axonal branches the high molecular weight proteins are transported at the highest rate, medium weights slower and low molecular weight proteins slowest. More proteins of high and low molecular weights are transported into the dorsal root whereas more of those of medium molecular weight are transported towards the sciatic nerve.


[Hypoplasia ponto-neocerebellaris (author's transl)]. In an otherwise healthy family three male infants fell ill with microcephaly, hypotonus of the muscles, non-specific hyperkineses, seizures, and rapid mental deterioration. In addition to microdysplasia in the cerebral cortex the main neuropathological findings were the separation of the dentate nucleus into many islets, an atypical band of the inferior olives, and deficiency of the pontocerebellar fibers in the basal part of the pons. The hypoplasia of the areas involved can be distinguished from the degenerative process in the dégénérescence systematisé optico-cochleo-dentelé (Nyssen-van Bogaert).


Craniometaphysial dysplasia with leukoencephalopathy. A case report. A case of craniometaphysial dysplasia with extensive degeneration of the cerebral white matter is presented. The cortex of the swollen part of the long bones was quite thin and there was striking trabecular atrophy. Both the vault and base of the skull showed marked thickening and sclerosis, leaving no interlaminal zone. It is probable that the diffuse degenerative change of the cerebral white matter with gliosis bears some resemblance to that produced by a circulatory disturbance of the great vein of Galen. Due to the narrowed foramen magnum, deformed atlase and axis, and the surrounding postoperative scar, the upper cervical cord was compressed, markedly atrophic and degenerated. Other segments of the cervical and thoracic cord displayed secondary wallerian degeneration and focal neurolytic lesions in the white matter. At the level of Th11 there was a pencil-like malacic lesion, suggesting an apparent interference of circulatory disturbance due possibly to the deformed vertebral column.


Changes in nerve and muscle produced by long-term nerve ligation. The long-term effects of constricting ligatures applied to the sciatic nerves of neonatal rats were examined. The nerves showed a decrease in diameter below the ligature but no accumulation of either SDH or AChE. The muscle innervated showed predominately high levels of SDH, mATPase, phosphorylase and glycogen. Following removal of the ligatures, the nerves were similar in size above and below the ligature, and the corresponding muscles showed an increased number of fibers with decreased SDH activity. Although mATPase, PAS and phosphorylase activities remained high, groups of lightly staining fibers were evident with the mATPase reaction. Long-term nerve ligation appears to produce changes in axonal size rather than in the fast component of axoplastic flow. Furthermore, the changes produced in muscle histochemistry by nerve ligation are closely related to changes in axonal size than to disturbances in flow.


Computed tomography for neurological intensive care patients. Report on one hundred CT examinations. The first 100 computed tomographic (CT) examinations of the patients on the neurological intensive care ward are discussed and reported on the basis of selected typical findings. Characteristic patterns of the CT findings in determined cerebral diseases are explained. The possibility and necessity of CT observations of the development, of inflammatory and cerebrovascular processes in particular are emphasized. A comparison of our experience with CT and other neuroradiological methods, is made. The clinical diagnoses, including the respective number of cases and the pertinent CT findings, are presented in a Table.


An overhead projection aid for teaching the principle of occlusion. A technique has been presented whereby models which can be animated are used to form a projected image on a screen and thereby demonstrate the principles of occlusion together with principles of articulators. The system can be used alone or in conjunction with clinical slides interjected at relevant places in the discussion. This technique has been found to be a very useful aid in the teaching of occlusion to both graduate and undergraduate students.


True hepatic teratoma with high alpha fetoprotein in serum. True hepatic teratoma is very rare, and only 23 cases including 7 in adults have been reported in the literature. In this report a case of true hepatic teratoma producing high alpha fetoprotein is presented.


Chelates of dicumarol I: Preparation and structure identification of magnesium chelate. A magnesium chelate of dicumarol was prepared by reacting a suspension of dicumarol and magnesium oxide in 50% water-methanol. GLC, thermogravimetric, and elemental analyses showed that this compound has a 2:1 ligand-metal stoichiometry with 2 moles of water associated with the complex. Although the chelate does not melt, two endothermic peaks at 205 and 274 degrees were observed in the thermogram, in contrast to a single endothermic peak corresponding to a melting point of 288 degrees for dicumarol. IR spectroscopy indicated that the magnesium is bonded between the carbonyl at C-2 and the oxygen at C-4' (or vice versa).


Selective photometric determination of betamethasone benzoate and other 21-hydroxycorticosteroids. Condensation of the glyoxal obtained by cupric acetate oxidation of 21-hydroxycorticosteroids with acetous phenylhydrazine reagent affords a near UV chromophore. All of the tested corticosteroids, including triamicinolone acetonide, which gives low color yields in the Porter-Silber reaction and its Lewbart-Mattox modification, gave similar absorption maxima (362-370 nm) and molar absorptivities (epsilon = 17,000-20,500). Since corticosteroid 21-esters and oxidation products do not undergo the reaction, the assay method based on it is stability indicating for betamethasone benzoate and the other test compounds. Procedures are described for the assay of two topical betamethasone benzoate preparations and hydrocortisone and prednisolone tablets; recovery and precision data are given.


Maternal serum alpha-fetoprotein in abnormal pregnancies and during induced abortion. Serial maternal serum alpha-fetoprotein (AFP) levels were determined by radioimmunoassay in patients with abnormal pregnancies and after saline-induced abortion. Results from patients presenting with mild or severe preeclampsia, fetal growth retardation and severe diabetes did not differ from the normal range. However, in patients with premature labor, the majority of AFP levels were significantly below the normal range, and peak AFP levels were achieved approximately one month earlier than normal. In patients whose pregnancies were terminated by abortion, the AFP levels exhibited a significant rise within a few hours after induction because of resorption of fetal elements into the maternal circulation.


Acridine orange-DNA complex in actinic keratosis. Electron microscopy processing and staining of nuclei were used to localize reaction products of acridine orange staining in actinic keratosis of human skin. Electron-dense granules about 10-100 nm in diameter were seen exclusively in the euchromatin portion of the nucleus. Almost all tumor cells had granules (mean = 65; SD = 26). These granules were also occasionally observed in the dermal connective tissue cells in the lesion. However, the mean number of 10 granules seen in these cells was definitely less than that of tumor cells. Normal skin controls did not have granules except occasionally in the basal cells of the epidermis.


Extensive in vitro transcription of rous sarcoma virus RNA by avian myeloblastosis virus DNA polymerase and concurrent activation of the associated RNase H. Conditions are described that promote the efficient reverse transcription of most of Rous sarcoma virus (RSV) RNA sequences by avian myeloblastosis virus DNA polymerase in vitro. A detailed analysis of the reverse transcription reaction was carried out using two procedures: in situ analysis of the RNA sequences transcribed and DNA-RNA annealing studies. Under optimal conditions, after 1 h of reaction, practically all RSV RNA sequences were transcribed with a frequency varying from 30 to 90%. The DNA product was at least 95% single stranded, had a chain length ranging from a few hundred up to 5,000 necleotide residues, half of it being larger than 1,000 residues, and, after hybridization at RNA excess, protected the entire RSV genome from RNase digestion, as monitored by the large T1 oligonucleotides of RSV RNA. Analysis of the product of a very short reaction time (5 min) showed that DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA. This in in agreement with our previous analysis of a much less efficient reverse transcription reaction. Under optimal conditions of reverse transcription, we find now that the RNase H associated with the avian myeloblastosis virus DNA polymerase is active in degrading the RNA moiety of the RNA-DNA hybrids synthesized.


Community-acquired Acinetobacter calcoaceticus var anitratus pneumonia. Two patients had community-acquired Acinetobacter calcoaceticus var anitratus pneumonia. Both patients were alcoholic and one was cirrhotic. One patient died and the other received two weeks of gentamicin therapy and survived. Misinterpretation of the sputum Gram stain delayed diagnosis and institution of proper therapy in both cases. In addition to organisms sensitive to penicillins such as Neisseria or Haemophilus, Acinetobacter must be considered in the differential diagnosis of community-acquired Gram-negative coccobacillary pneumonia.


Marrow iron.

Marrow iron. Marrow aspirates stained with the Prussian blue reaction give a variety of patterns. Characteristic pictures are shown for megaloblastic anemia, infection, and subjects after injection of iron dextran. The appearance of iron in histiocytes in marrows stained with Wright's stain is differentiated from noniron deposits. A graph shows the relative distribution of iron in RBCs and storage in different clinical situations.


[Study of low density lipoproteins in hyperlipoproteinemia by the method of double immunodiffusion]. By way of gel double immunodiffusion a certain heterogeneity of low density lipoproteins was observed to manifest itself in an additional band of immunoprecipitation. The incidence of this additional band does not exceed 1/3 of the cases of low density lipoproteins studies in normal individuals, while in those with ischaemic heart disease the additional band is found twice-thrice as often, the highest incidence being noted in patients with ischaemic heart disease and Type IIb hyperlipoproteinemia (93% of cases). The revealed immunochemical heterogeneity of low density lipoproteins was shown to be not connected with the appearance of any new, additional antigen in their structure. Most probably it is attributable to the presence of lipoprotein particles with a quantitatively different protein and lipid composition, probably of intermediate lipoprotein metabolites in blood plasma, or conformation changes in the structure of low density lipoproteins.


[Cellular immunity in Sjögren's syndrom (author's transl)]. The authors made cellular immunological investigations with lacrymal and salivary gland antigens on 11 Sjögren's syndrome patients. The changes following the immuno-suppressive treatment were observed by the blasttransformation of the lymphocytes and by the inhibition of the leuckocyte migration.


[Alpha1-fetoprotein: physiology, pathology and diagnosis especially in childhood (author's transl)]. Alpha1-fetoprotein (AFP) is an alpha1-glycoprotein which can be found in high concentration during fetal development in many mammals, birds, sharks and, also, man. The alpha-fetoproteins of various species have similar physico-chemical properties and often common antigenic determinants. Differences of microheterogeneity depend on a different content of sialin-acid. During human fetal development the serum AFP concentration falls with increasing gestational age. 4-5 weeks after birth AFP can be detected usually in low serum concentrations. Using more sensitive immunulogic techniques e.g. radioimmunoassay there was shown that AFP is present in sera of normal adults in concentrations of 10-20 ng/ml. AFP serum concentrations rise physiologically during pregnancy up to 500-550 ng/ml. During fetal development liver, yolk sac and gastrointestinal tract are the major sites of synthesis. In primary liver cell carcinoma, hepatoblastoma and in teratoblastoma containing yolk sac tissue AFP synthesis rises in tumor cells; the AFP serum concentration increases above 2 microgram/ml. In patients with benign liver diseases e.g. virus hepatitis, a transient rise of AFP serum concentrations was seen. Moreover, increased levels of AFP were found in hereditary diseases e.g. congenital tyrosinemia, ataxia-telangiectasia and in the amniotic fluid in congenital nephrosis of Finnish type. AFP assay in serum is clinically important for the control of course and treatment of primary liver cell carcinoma and teratoblastoma. AFP assay in amniotic fluid is a method for the prenatal detection of neural tube defects and the fetal distress syndrome, especially.


[Serum concentrations of proteinase inhibitors, complement components and of acid alpha-1-glucoprotein in children with myocarditis (author's transl)]. We examined 4 panels of children and 40 control patients for their serum levels of the complement components C3, C4 and the C3 activator, the proteaseinhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and acid alpha-1-glycoprotein. Children of the group with active myocarditis revealed the consumption of the complement system, increased protease inhibitors and elevated acid alpha-1-glycoprotein. Group 2, children with the clinical diagnosis chronic myocarditis or status post myocarditis showed in six of seven cases low complement levels and elevated alsGP. 4 children showed increased A1AT and five increased A2MG. In the third panel: status post myocarditis, we estimated in five of eight patients complement activation, 4 children showed increased A2MG and alsGP and in 3 cases elevated A1AT levels were detected. Group 4 children revealed no complement consumption and showed no increased levels for the other proteins estimated, with the only exception of 1 case with increased alsGP. The children of the control group showed normal levels for the six proteins. By means of the examinations an inflammatory process can be detected, tissue injury can be indicated and the participation of the immune system can be shown.


[Immunohistochemical and radioimmunological demonstration of alpha1-fetoprotein in nonmalignant changes of human gastric mucosa (author's transl)]. The occurrence of alpha1-fetoprotein in nonmalignant changes of the gastric mucosa was investigated by means of immunohistochemistry and radioimmunoassay. The investigations were performed in tissue sections, cytological imprint preparations as well as in homogenized tissue samples (obtained by gastroscopy). alpha1-fetoprotein could be demonstrated by immunohistochemistry in about 90% of the samples originating from the surroundings of gastric ulcer and the region of gastrojejunostomy after B II-resection. The RIA was positive in about 75% of the tissue samples, whereas from gastric juice only 40% of positive results could be obtained. No alpha1-fetoprotein-activity could be demonstrated in serum samples. These investigations indicate that alpha1-fetoprotein is not exclusively synthesized by embryonic or neoplastic tissues and also can be synthesized also by regenerating cell-systems. It may be supposed that this synthesis represents an unspecific answer to growth-stimulation.


Recent advances in microbiology in leprosy. The recent advances in microbiology of leprosy are reviewed. Till now the leprosy bacillus had not been cultivated in laboratory media; the recent claims of success have not been confirmed. There has been a breakthrough in the experimental transmission of leprosy to experimental animals--the white mice, the immune depressed white mice, and the nine-banded armadillo. Apart from providing definite proof for the causative relationship of the bacillus discovered by Hansen and the disease leprosy, the experimental transmission to animals have considerably advances our knowledge about the disease. The mouse has provided a suitable model for screening of antileprosy drugs, detecting development of drug resistance, ascertaining viability of the bacilli and determining the genuineness of a supposed culture of the leprosy bacillus. The armadillo has provided a model for making basic studies of the disease as it occurs in man. Further, the generalised infection in armadillo has provided large amounts of leprosy bacilli for preparing standardised lepromin, for preparing a specific skin-testing antigen containing the active protein fraction of the bacillus, and a step towards the production of a vaccine against leprosy. It is interesting to note that the fact that the protein fraction of the bacillus was responsible for the positive lepromin reaction was discovered by Dharmendra far back in 1941.


The Student Response System. A 5-year Mayo Medical School Experience. The Student Response System is a commercially available electronic communication system applicable to many teaching and testing situations. At rather short intervals during a programmed course, the instructor projects on a screen multiple-choice questions that the student should have learned to answer during one or two previous teaching steps. Correct and incorrect responses are indicated on a panel on the desk of the instructor. If students fail to give the correct answer, the teaching step is repeated. Recurrent incorrect answers indicate that explanations had been inappropriate or that test slides were poorly designed. Thus, the system allows the instructor to monitor his own performance. Students are kept alert and attention spans are prolonged; communication is increased and personal embarrassment is minimized. Only the instructor has access to the individual student's response. Most learning takes place in the classroom rather than during subsequent private study. This system is quite versatile and offers potential uses hitherto untested in medical education at all levels.


Dissolution of cholesterol gallstones by ursodeoxycholic acid. 44 patients with radiolucent gallstones in gallbladders visible on cholecystography were randomly alloted to three treatment groups: ursodeoxycholic acid (600 mg/day), ursodeoxycholic acid (150 mg/day), a placebo. At the end of six months' treatment, cholecystograms of all the patients were interpreted by radiologists who were not aware of the treatment. Dissolution of gallstones occurred in 8 (26%) of the 31 patients treated with ursodeoxycholic acid, but not in the placebo group. Ursodeoxycholic acid had no hepatotoxicity, as assessed by standard liver-function tests. These results indicate that ursodeoxycholic acid, the 7beta epimer of chenodeoxycholic acid, is effective in the dissolution of cholesterol gallstones.


Fibrinolytic response to surgery. Labile and stable patterns and their relevance to post-operative deep venous thrombosis. Fibrinolytic activity after elective abdominal surgery was studied in 53 middle-aged cancer-free patients. The pattern of their response demonstrated both stable and labile populations. In 10 of the 13 patients in whom fibrinolytic activity fell rapidly to below 5-6 units, deep venous thrombosis (D.V.T.) developed on the first postoperative day. This suggests that reduced fibrinolytic activity after operation is involved in the establishment of postoperative D.V.T. and that the incidence of early thrombosis could be reduced by enhancement of postoperative levels of fibrinolytic activity.


Low levels of eosinophil cationic proteins in patients with asthma. Arginine-rich cationic proteins are a major constituent of the granules of eosinophil leucocytes. In healthy individuals, serum levels of cationic proteins were correlated with blood eosinophil-counts. In asthma patients blood eosinophil-counts were raised but serum cationic-protein levels were lower than expected. Some patients with intrinsic asthma had very low cationic-protein levels despite high eosinophilia. A challenge test in one patient caused a striking decrease in serum cationic proteins but no change in eosinophil-count. Assays of serum "eosinophil" cationic proteins may be useful in the differentiation of atopic and intrinsic asthma and may throw light on pathogenesis.


beta-adrenergic agonist resistance in normal human airways. The response to a beta2-agonist (salbutamol) was assessed by measuring specific airways conductance (sGaw) in healthy volunteers following increasing doses of inhaled or intravenous salbutamol. Regular inhaled salbutamol in doses exceeding 200 microgram four times a day produced a progressive loss of airways responsiveness to both inhaled and intravenous salbutamol (beta-agonist resistance). In salbutamol-resistant subjects full bronchodilator sensitivity was restored 3-5 h after intravenous hydrocortisone.


Pattern of maternal F-cell production during pregnancy. Maternal haemoglobin F production has been monitored in 11 normal pregnancies, with an immunofluorescent technique. In all cases there was a significant increase in the number of F-cells which reached a peak at 18-22 weeks' gestation. Comparison of the numbers of F-cells with the percentage haemoglobin F determined chemically indicated that the increase in maternal haemoglobin F synthesis results from an increased production of F-cells rather than from an increased synthesis of haemoglobin F by the F-cells. There was a highly significant increase in the number of F-cells in a pregnant beta-thalassaemia heterozygote.


Defective neutrophil migration in sarcoidosis. A quantitative skin-window-chamber technique was used to study 13 patients with sarcoidosis. Neutrophil migration into the chambers in 24 hours was less than that in normal subjects. Chemotaxis of neutrophils in vitro was normal. Serum from patients with sarcoidosis inhibited neutrophil chemotaxis more than normal serum.


Levamisole in the treatment of Crohn's disease. In 8 patients with active uncomplicated Crohn's disease who were not receiving any specific therapy, a clinical remission was induced with an elemental diet, and then an attempt was made to maintain this remission with levamisole. This regimen resulted in excellent control of the disease. A drug-induced arthritis developed in two patients and resolved spontaneously. There were no other significant side-effects.


Alanine inhibition of pyruvate kinase in gliomas and meningiomas. A diagnostic tool in surgery for gliomas? In gliomas a shift was found in the composition of enzymes, manifested by abnormal inhibition of pyruvate kinase by alanine. This study demonstrates the appearance of the MII-type isoenzyme in various gliomas. This MII type is also found in meningiomas but not in normal brain tissue. The method of enzyme examination described may be valuable as a diagnostic aid in the surgery of gliomas.


Wheat fibre and irritable bowel syndrome. A controlled trial. Twenty-six patients with irritable bowel syndrome entered a controlled trial of diets with a high or low wheat-fibre content. After 6 weeks on the high-wheat fibre regimen there was significant improvement in symptoms and an objective change in colonic motor activity. No such improvement occurred on the low-fibre regimen. Patients with irritable bowel syndrome should be encouraged to increase their daily intake of wheat fibre.


Influence of sex on histocompatibility matching in renal transplantation. Histocompatibility matching for the HLA-A and HLA-B loci specificities influenced the survival of cadaver kidney transplants in male recipients but not in female recipients. The correlation between HLA matching and transplant survival for a three-year follow-up period was highly significant for 3455 male recipients but not significant for 2007 female recipients. Since this effect was not dependent on the sex of the donor, a sex-associated histocompatibility antigen could not have been responsible. The reason why histocompatibility matching influences one sex and not the other is not known. The observation may have improtant practical consequences and may help to explain difficulties in establishing a strong correlation between HLA matching and cadaver transplant outcome.


Evidence for dopaminergic control of thyrotrophin secretion in man. After administration of the dopamine-receptor-blocking drug, metoclopramide (10 mg orally), there is significant release of thyrotrophin (T.S.H.) in hypothyroid patients which is not evident in euthyroid subjects. This is not due to spontaneous fluctuation in basal T.S.H. levels, and it indicates inhibitory dopaminergic control of T.S.H. release in man. The lack of significant T.S.H. release in euthyroid subjects may be due to the inhibitory effects of normal circulating levels of T3 and T4 on T.S.H. release. The T.S.H. response in hypothyroidism is significantly correlated with both T3 and T4 levels, suggesting suppression of this inhibitory pathway in increasingly severe hypothyroidism.


Impaired prolactin release in Huntington's chorea. Evidence for dopaminergic excess. Eight individuals with Huntington's chorea had low basal and impaired human prolactin responses to both chlorpromazine and thyrotrophin-releasing hormone. These findings are compatible with enhanced hypothalamic dopaminergic activity in this disease. Two juvenile rigid patients with Huntington's chorea had excessive prolactin responses to chlorpromazine. Approximately half of the twenty-three potentially affected first-degree relatives of patients with Huntington's chorea had normal prolactin responses to chlorpromazine. However, twelve had significantly abnormal responses-seven in one direction, and five in the other. The predictive value of these findings in terms of presymptomatic diagnosis will be revealed by a longitudinal study. A biochemical method for the early recognition of the condition would have profound implications for genetic counselling.


Familial islet-cell adenomatosis.

Familial islet-cell adenomatosis. Familial multiple adenoma of pancreatic beta-cells is described for the first time. The occurrence of diabetes mellitus in different members of the family raises the possibility of a common genetic origin for the multiple islet-cell adenomas and the diabetic trait. The evidence suggests that this gene is autosomal and dominant, that it is not linked with the HLA antigens, and that is causes an abnormal sensitivity of the beta-cells, which become hyperplastic or hypofunctional.


Plasma-arginine-vasopressin response to insulin-induced hypoglycaemia. Insulin-induced hypoglycaemia caused a threefold rise in plasma-arginine-vasopressin concentration (to 4-36 +/- 0-77 pmol/1) in ten subjects who had normal posterior-pituitary function. Plasma-arginine vasopressin reached a peak 30 min after injection of insulin. Plasma concentrations of arginine vasopressin obtained with hypoglycaemia were similar to those achieved after overnight dehydration for 14-16 h. No rise in plasma-arginine-vasopressin was observed in three patients with cranial diabetes insipidus in whom severe hypoglycaemia developed after insulin infusion. It is suggested that the measurement of arginine vasopressin during insulin-induced hypoglycaemia may be a useful clinical test of posterior-pituitary function.


Inhibiton of Fc-rosette formation by serum of patients with renal allograft rejection. A test which measures the inhibition of Fc-rosette formation by the patient's serum was used in a group of 23 renal allograft recipients. All 13 serum samples obtained during acute rejection and 2 of 3 obtained during chronic rejection showed strong inhibitory activity, suggesting that the test is of value in confirming the presence of rejection. Its value as a predictive test remains to be established. Preliminary experiments suggest that both immune complexes and alloantibodies contribute to the observed inhibition of Fc-rosette formation.


Treatment of paracetamol (acetaminophen) poisoning with N-acetylcysteine. Fifteen patients with paracetamol (acetaminophen) poisoning were treated with intravenous N-acetylcystein (300 mg/kg given over 20 h). Mean admission and 4 h plasma-paracetamol concentrations were 262 and 369 microgram/ml, respectively. Liver-function tests remained normal or were only slightly disturbed in 11 of 12 patients treated within 10 h of paracetamol ingestion. Severe liver damage developed in the other patient and in the three in whom treatment was started more than 10 h after paracetamol ingestion. In contrast to cysteamine, N-acetylcysteine was very well tolerated and has the advantage of being available as a pharmaceutical preparation in a 20% sterile solution.


Possible effects of growth hormone on development of acute lymphoblastic leukaemia. Growth hormone (G.H.) or a G.H.-dependent somatomedin may be involved in the process of acute lymphoblastic leukaemia (A.L.L.). Growth hormone has a trophic effect on lymphoid tissue and also specific receptors on lymphocytes, most probably T cells. Hypophycess. Resting concentrations of G.H. and somatomedin activity are raised in some children with A.L.L. and may be reduced after remission is achieved. It is suggested that control of G.H. and/or somatomedin concentrations may be necessary for adequate treatment of some cases of A.L.L. in children.


Is there a scientific basis for six-monthly dental examinations? This review of the need for 6-monthly dental checks was limited to persons aged 12 years and more, because there are no data on the rates of progression of diseases of the primary dentition. Dental caries in the permanent teeth is a very slow disease, and takes about 2 years to progress through the enamel. Persons having dental examinations at intervals longer than 6 months were not at a disadvantage. They did not have more severe dental caries or periodontal disease than those attending at intervals of 6 months. Neither was there any evidence that an interval of more than 6 months would affect the prognosis for orthodontic treatment. Oral malignancies are rare in British populations, and routine 6-monthly screening to detect oral malignancies is unreasonable.


Gentamicin-resistant Klebsiella aerogenes in a urological ward. A gentamicin-resistant strain of Klebsiella aerogenes was isolated from the urine of 17 patients out of 237 admitted to a male urological ward between Jan. 21 and May 9, 1977. The factors most frequently associated with K. aerogenes in the urine were catheterisation and antibiotic therapy. Often the epidemic strain (type K16) was found not only in the patients' faeces but also on more remote skin sites such as hands, knees, groins, and the umbilicus. Resistance to gentamicin and many other antibiotics was R-factor mediated. Barrier nursing of colonised patients, stringent staff handwashing with chlorhexidine, and the use of disposable aprons seemed to contain the outbreak. Hand carriage was demonstrated in one nurse 62 days after she had left the ward.


The metabolic homoeostatic role of muscle and its function as a store of protein. Evidence is produced that the skeletal muscles of the body, which weigh 21 times as much as the liver, form a major store for protein and act as a great metabolic regulatory organ which helps to maintain acceptalbe levels of aminoacids and glucose in the circulation. This concept has relevance to various diseases.


Autoantibody against B lymphocytes.

Autoantibody against B lymphocytes. The sera of 20 out of 99 (20%) normal males and females were shown to have antibodies that kill their own B lymphocytes. The sera were specifically cytotoxic to B lymphocytes and not T lymphocytes. Allogeneic B lymphocytes were also killed by these autocytotoxins. Maximum killing of B lymphocytes occurred when the incubation temperature in serum was 5 degrees C and complement incubation temperature was 20 degrees C. Under these cold conditions, some sera reacted up to a dilution of 1/16. These results suggest that positive B-lymphocyte crossmatches found in kidney transplantation do not detect alloantibodies. Since the B-cell autoantibodies tend to appear at the time of immunisation, their role as autoregulators of the antibody response is an intriguing possibility.


Mucosal changes in the gastric stump 20-25 years after partial gastrectomy. Out of 421 patients who had partial gastrectomy 20-25 years ago for gastric or duodenal ulcer, 108 were examined by endoscopy with multiple biopsy. In no case were the endoscopic appearances of the mucosae interpreted as malignant, though in 2 patients the clinical history suggest malignant disease. Histological examination revealed infiltrating carcinoma in 4 patients, 3 of whom had intramucosal carcinoma only. 3 further patients had severe dysplasia (carcinoma-in-situ). Only 1 patient had a near-normal mucosa close to the anastomosis; in the remainder the gastric remnant showed various degress of dysplasia, metaplasia, or chronic atrophic gastritis. In the patients with carcinoma only 28 (12%) of the 226 biopsy specimens revealed the malignant lesion. Patients who have had partial gastrectomy for benign lesions are at high risk of gastric-stump carcinoma. If, 20 years after operation, they have an endoscopy with multiple biopsy, stump carcinoma may be detected early when the prognosis after operation is probably good.


Influence of lipid concentrations and age on transfer of plasma lipoprotein into human arterial intima. Transfer of low-density lipoprotein (L.D.L.2) from plasma to arterial intima was studied in 16 patients undergoing arterial surgery. Autologous labelled lipoprotein was used to demonstrate that L.D.L.2 enters the intima from plasma. Net flux of L.D.L.2 appeared to increase with age. Within each age-group the net flux of L.D.L.2 showed a pronounced positive correlation with plasma-L.D.L.-cholesterol concentrations. This may account in part for the association between hypercholesterolaemia and the development of atherosclerosis.