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Serum level of pregnancy associated alpha2-globulin in patients with spontaneous abortions.

Pregnancy associated alpha2-globulin serum levels have been measured in abortion-cases. In cases of incomplete abortion with placental destruction and missed abortion this alpha2-globulin level significantly decreased, but no changes could be found when the placenta remained intact. The prognostic value of this pregnancy protein is discussed.

Sequences related to the RNA tumor viruses in the RNA and DNA of human leukemias and lymphomas.

DNA-RNA hybridization was used to explore whether human neoplasias contain RNA molecules having sequence homologies to those of the RNA tumor viruses known to cause similar diseases in animals. The pattern of specific RNAs found in the human tumors showed a remarkable concordance with the predictions deducible from the animal systems. Thus human breast cancer contains RNA homologous only to that of the murine mammary tumor virus (MMTV). Human leukemias, sarcomas, and lymphomas (including Hodgkin's and Burkitt's) all contain RNA with sequence homology to the murine leukemia virus (RLV) and not to MMTV RNA. Finally, as in the case of the mouse, none of the human tumors examined contain RNA related in sequence to that of the avian myeloblastosis virus (AMV). The RNA detected in all of the human neoplasias was demonstrated to be of high molecular weight (1 times 10(7) daltons) and encapsulated with a reverse transcriptase in particles having densities between 1.16-1.19 g/ml. Further, the RNA of these human tumor particles was related in sequence to the murine viruses that cause the corresponding neoplasias in mice. Thus, 4 features diagnostic for the murine oncogenic viruses are satisfied by the particles found in the human cancers. Finally, it was shown by "recycling" experiments that the DNA from human leukemic cells and from lymphomatous tissue contained particle-related sequences that could not be detected in normal DNA. This finding was further substantiated by studies with identical twins in which it was shown that the leukemic twin contained particle-related sequences that could not be detected in the leukocytes of his identical healthy sibling. These findings are inconsistent with hypotheses that require chromosomal transmission in the germ line of complete copies of the information required to produce malignancy and the associated virus particles.

Morphological, chemical, and antigenic organization of mammalian C-type viruses.

New features in the architecture of mammalian type C viruses, in particular knoblike surface projections and hexagonally arranged subunits on the core shell could be demonstrated by electron microscopy, taking advantage of newly developed preparation techniques. As examples, murine leukemia viruses (MuLVs) and newly isolated porcine and bovine C viruses are presented. The major proteins of a MuLV were isolated and partially characterized in chemical terms and with respect to their serological and other biological activities, such as interfering and hemagglutinating (HA) capacity. Most of the characterized proteins could be localized in particular substructures of the virion either by selective removal or isolation of electron microscopically identifiable constituents. The information obtained allowed the design of a more detailed model of mammalian C viruses. Special attention was devoted to the further characterization of interspecies antigens of mammalian C viruses. Different antigenic determinants were revealed. Their distribution allows further subgrouping of mammalian C viruses.

[Effects of amantadine on heart and circulation].

The effect of amantadine-hydrochloride on heart and circulation is studied in 7 anesthetized, otherwise normal dogs with a mean body weight of 27.2 kg and in 8 heart-lung preparations of dogs. Arterial blood pressure, right atrial pressure, cardiac output and heart rate are measured and continuously monitored. Stroke volume and peripheral resistance are calculated. Left ventricular peak- and enddiastolic pressure, the rate of rise of intraventricular pressure and t-dp/dt are additionally measured in the heart-lung-preparations. Below 3 mg with kg(-1) in anesthetized dogs and 10 mg in the heart-lung preparation, respectively, a positive inotropic effect of amantadine is observed. This effect is caused by a liberation of catecholamines. Higher dosage of amantadine decreases cardiac contractility significantly. Therefore the negative inotropic influence of the drug itself has to be distinguished from the indirect sympathomimetic effect resulting from local release of myocardial catecholamines. Cardiac arrhythmias which occur in several experiments, can mostly be eliminated with propranolol or other drugs like lidocaine or sparteinsulphate.

Deoxycytidine kinase: properties of the enzyme from human leukemic granulocytes.

Deoxycytidine kinase, which phosphorylates deoxycytidine (CdR) and its analog, cytosine arabinoside (ara-C), has been purified 71-fold from human leukemic cells. Biochemical properties of the partially purified enzyme included a molecular weight of 68,000, Kms of 7.8 muM for CdR and 25.6 muM for ara-C, and optimal activity with ATP and GTP as phosphate donors. Ara-C phosphorylation was strongly inhibited by CdR (Ki = 0.17 muM) and dCTP (Ki = 7.3 muM) and was weakly inhibited by ara-CTP (Ki = 0.13 mM). Purification by calcium phosphate gel elution and DEAE chromatography effectively separated this enzyme from cytidine deaminase, which deaminates both CdR and ara-C, and from uridine-cytidine kinase, the enzyme which phosphorylates 5-azacytidine. CdR kinase activity was found to decrease and cytidine deaminase to increase with maturation of normal and leukemic granulocytes. Myeloblasts purified by Ficoll sedimentation revealed an average kinase activity of 15.4 U/mg protein in acute myelocytic leukemia and 12.3 U/mg protein in blastic crisis of chronic myelocytic leukemia (CML). The average ratio of CdR kinase to deaminase activity in crude cell extracts varied from 0.197 in AML and 0.089 in blastic crisis to 0.0004 in normal granulocytes, reflecting the changes which take place with cellular maturation. The absolute levels of kinase and deaminase and the ratio of these two enzymes varied considerably among patients with AML, indicating that quantitative differences may be found in the metabolism of CdR and its analogs in leukemic cells.

The localization of the basic protein and N-2 in diseased myelin.

A non-penetrating reagent 4,4'-diisothiocyano-2,2'-ditritiostilbene disulfonic acid ([3H]DIDS) has been used to label isolated normal and diseased myelin. The basic protein and the hydrophobic protein, N-2, were isolated from each myelin. When normal myelin was labeled the specific activity of the basic protein was about 25% of that of the hydrophobic protein (N-2). The specific activities of these two proteins isolated from chronic multiple sclerosis myelin were similar to those of the normal myelin. In contrast, the specific acitivity of the basic protein isolated from acute multiple sclerosis myelin was about 400% higher than that of the basic protein isolated from either normal or chronic multiple sclerosis myelins. The specific activity of the N-2 protein was only 50% of that of the N-2 protein isolated from normal and chronic multiple sclerosis myelins. It was concluded that the arrangement of proteins in isolated chronic multiple scerosis myelin was not markedly altered in comparison to that of isolated normal myelin. However, the arrangement of proteins in acute multiple sclerosis myelin appeared to be considerably different from that of the other two myelins.

A scanning electron microscope study of human cerebral arteries.

Human cerebral arteries were obtained from autopsy, fixed under pressure, cut open, and tacked onto pieces of cork. For one artery the intima was partly teased away, exposing the media, and treated with a silver nitrate process. For another artery the adventitia was exposed. Both arteries were processed through graded ethanols and coated with gold paladium for the scanning electron microscope. The collagen fibers of the adventitia were approximately 5 mum in diameter and consisted of a bundle of microfilaments, each of which had a diameter of 800-1000 A (1 A = 10(-10) m). The collagen fibers were oriented parallel to the long axis of the artery. The muscle cells of the media had a diameter of 2-5 mum and were arranged circumferentially with a pitch of approximately 20 degrees. The collagen fibers of the media travel perpendicular to the muscle cells, and parallel to the long axis of the artery. The fibrillar components of the elastin in the intima had a diameter of approximately 700-1000 A and were arranged parallel to the long axis of the artery. It was postulated that the fibrillar part of the elastin was the elastic component of the elastin.

Effects of barbiturates on ultrastructure and polymerization of microtubules in vitro.

Barbiturates were examined for in vitro effects on ultrastructure of the frog sciatic system and polymerization of microtubules (MT) in a brain supernatant. Exposure for 5-17 h to 2.0 mM barbiturates caused a considerable loss of MT in ganglionic cell bodies and sciatic axons. This was mostly followed by a proliferation of 10 nm filaments. Under similar conditions treatment with 1 mM NaCN or 0.1 mM 2,4-DNP did not change the number or ultrastructure of MT and filaments. Eight barbiturates, varying in binding ratios to serum albumin and partition coefficients, were tested for effects on polymerization of MT using viscometry. Inhibitory effects were found which correlated with their reported ability to bind to albumin and brain fractions. Dimethylsulphoxide and ethanol were used as solvents for some of the barbiturates. These solvents at 1% had stabilizing effects on MT. The present results are discussed in relation to previous findings of inhibition of rapid axonal transport in vitro in the frog sciatic system by barbiturates.

Mechanisms of chromosome banding. VII. Interaction of methylene blue with DNA and chromatin.

The binding of methylene blue to DNA and chromatin treated in various ways was examined by equilibrium dialysis. The maximum r value (moles of bound dye/mole of nucleotide) was 1.0 for DNA, 0.6 for unfixed chromatin, and 0.83 for chromatin fixed in methanol-acetic acid. When fixed chromatin was treated with saline-citrate at 60 degrees C for 3 hours, as used for G-banding chromosomes, the r value decreased from 0.83 to 0.55. When unfixed chromatin was treated as for R-banding the r values also dropped. Equilibrium dialysis indicated there was no disproportionate increase of dye binding as the concentration of DNA increased. -- These results, and others, suggest that some of the Giemsa negative regions of G- and R-banded chromosomes are due to the denaturation of non-histone proteins so that they more effectively cover the DNA and prevent side binding of the thiazin dyes.

Differential effect of task relevance on early and late components of cortical and subcortical somatic evoked potentials in man.

The effect of "task relevance" on early and late components of cortical and subcortical somatic evoked potentials (SEPs) was studied in a group of Parkinsonian patients operated on under local anesthesia for treatment of prominent unilateral tremor. 1. SEPs produced by median nerve stimulation were found at contralateral cortical (ss), thalamic (vcpci, vcai), lemniscal (Lm), postilemniscal (PoLm), prelemniscal (Raprl) and reticular (Ttc) regions. No SEPs were found in other contiguous thalamic (M,Pf, ce) and subthalamic (Q) regions. 2. Subcortical early SEP components consisted of two monophasic positive potentials distributed within a circumscribed thalamo-lemniscal region where electrical stimulation elicited consistent sensory responses circumscribed to contralateral hand and face. In contrast, subcortical late SEP components consisted of monophasic or polyphasic, positive or negative potentials distributed in a widespread, thalamic, lemniscal, prelemniscal and reticular region where elecrical stimulation elecited sensory or motor responses of various types. Subcortical early and late SEP components appeared together in lemniscal, thalamic and cortical regions but they wers separated at postlemniscal (only early) and prelemniscal and reticular ones (only late). 3. Significant amplitude changes in cortical and subcortical late SEP componets were found concomitant to variations in "task relevance": they decreased when patients shifted from novelty to habituation, they increased when patients shifted from habituation to attention and they decreased when patients shifted from attention to distraction. In contrast, no significant ampiltude changes in cortical and subcortical early components were found when patients shifted through these various "task relevance" conditions.

A dual marking technique for microelectrode tracks and localization recording sites.

This paper describes techniques for marking both microelectrode tracks and exact recording loci using a combination of fast green dye and horseradish peroxidase (HRP). The procedure involves coating the exterior of HRP filled microelectrodes with fast green dye in order to identify electrode tracks, and ejecting HRP from the electrode to mark recording loci. Rapid, multiple marks can be made with this technique without harming the recording capabilities of the micropipette.

[Influence of hormonal contraceptives upon carbohydrate metabolism in women with abnormal glucose assimilation during previous pregnancy (author's transl)].

Carbohydrate metabolism was examined--by means of the 100-gm-glucose standard tolerance test--in 23 patients showing a decrease in glucose tolerance during pregnancy; the examinations were carried out postpartum and after a three months' intake of a hormonal contraceptive (compound preparation: mestranol 0.1 mg, lynestrenol 1.0 mg). The peak values and the two-hour-values, being statistically significant, showed a normalization after delivery, and furthermore again a deterioration of glucose tolerance during the intake of the preparation. A remarkable increase in weight, as a possible cause for the dysbolism during contraception, was not observed.

Alteration of the antibody response to Escherichia coli O antigen in mice by prior exposure to various somatic antigens.

In the present study in mice we used the Jerne plaque assay to compare the immunity enhancing potential of different Gram-negative bacteria with special regard to their endotoxin. The results confirm the recent finding that injection of Escherichia coli bacteria of various serotypes may enhance the IgG antibody response to the O antigen of a serologically unrelated E. coli strain injected subsequently, but may suppress the IgM antibody formation. The O antibodies formed were of low avidity but were antigen specific. Smaller amounts of antibodies were formed to a serologically unrelated antigen, E. coli O76, which had not been injected. Of the strains tested as primary stimuli E. coli O4 gave considerably greater enhancement than any other serotype including the homologous E. coli O6, when a short interval between the injections was used. The influence of O4 on the serologically unrelated anti-O6 response was stronger than on the response to the cross-reactive E. coli O18 antigen, suggesting that O antigen cross-reactivity is not the basis for the immunomodulation. Formalin-killed bacteria were more effective in this respect than boiled bacteria or purified lipopolysaccharide and rough mutants (E. coli R1--R4) and E. coli O4 were less effective than many of the other smooth E. coli. These findings suggest that shared determinants in the lipid, basic carbohydrate core or Kunin common antigen portions of the endotoxin do not play the major immunomodulating role in this system. Salmonella reading but not Pseudomonas aeruginosa affected the anti-E. coli O6 response in a similar manner. One explanation for the alterations in the immune response observed implies the presence of an antigen determinant shared by many Enterobacteriaceae in such a position in relation to the O antigen that it can be utilized for cellular co-operative events in the O antibody response. The protein portion of the endotoxin protein--lipid--carbohydrate complex is a possible location.

Studies of lymphocyte stimulation by intact tumor-cell and solubilized tumor antigen.

BALB/c mice were rendered immune to syngeneic SV40-induced sarcoma by subcutaneous injection of mKSA-TU5 tissue-culture adapted cells. Spleen cells from immune mice were examined for tumor-cell neutralization in the Winn assay as well as in in vitro lymphocyte stimulation assays. A microculture (200 mul) lymphocyte stimulation (LS) assay utilizing immune spleen cells was employed with mixed lymphocyte/tumor-cell cultures (MLTC) and the papain crude soluble (CS) extracts from mKSA-TU5 cells. Specificity in the LS assay was determined using spleen cells from mice immune to other syngeneic tumors and by soluble antigenic preparation of normal BALB/c spleen cells. The Winn assay studies demonstrated that spleen cells from mKSA-sensitized mice neutralized mKSA tumor cells and this was corroborated by their resistance to direct tumor challenge. Positive lymphocyte transformation responses in MLTC were observed when mKSA-TU5-sensitized spleen cells were mixed with mitocycin-C-treated intact tumor cells or when papain-solubilized antigens of mKSA cells were employed, but not with non-immune spleen cells or with a soluble antigen from normal cells. Papain-solubilized antigen preparations employed in in vitro assays also immunized against challenge with mKSA tumor cells. Specificity of these lymphocyte transformation reactions was demonstrated with non-sensitized lymphoid cells or lymphoid cells from mice sensitized with a syngeneic Kirsten virus-induced respond. Thus, mKSA tumor surface antigens were recognized on intact tumor cells or with microgram quantities of papain-solubilized extracts from these tumor cells. We believe the lymphocyte stimulation assay affords a method for demonstrating the presence of tumor-specific antigen and for monitoring further purification procedures.

Macromolecular synthesis in Streptomyces antibioticus: in vitro systems for aminoacylation and translation from young and old cells.

In vitro systems for the aminoacylation of transfer ribonucleic acid (tRNA) and for polypeptide synthesis have been constructed from young (12-h cultures, not producing actinomycin) and old (48-h cultures, producing actinomycin) cells of Streptomyces antibioticus. When Escherichia coli aminoacyl-tRNA synthetases were used to acylate S. antibioticus tRNA's, it was observed that, per absorbance unit of tRNA, the tRNA's from 48-h cells had a lower ability to accept the amino acids, leucine, serine, pheynlalanine, methionine, and valine than did the tRNA's from 12-h cells. Individual differences were observed between aminoacyl-tRNA synthetases from 12-h cells and those from 48-h cells with respect to the rate and extent of aminoacylation of E. coli tRNA with the five amino acids listed above. In vitro systems for the synthesis of polyphenylalanine have been constructed from 12- and 48-h cells. Ribsomes and soluble enzymes from 12-h cells are more efficient than those from 48-h cells in supporting polyphenylalanine synthesis, and, although the activity of both systems can be stimulated by the addition of E. coli tRNA, the higher level of incorporation observed in the unstimulated 12-h system (ribosomes and soluble enzymes) is maintained. Indeed, the difference in capacity for polyphenylalanine synthesis between in vitro systems from 12- and 48-h cells is greater when the systems are maximally stimulated by E. coli tRNA. Cross-mixing experiments reveal that enzymes from 48-h cells support a slightly higher level of polyphenylalanine synthesis than enzymes from 12-h cells with ribosomes from either cell type, and that the ribosomes are the primary agents responsible for the decreased efficiency of the in vito system from 48-h cells are compared with that from 12-h cells. To determine whether ribosome-associated factors were responsible for the relative inefficiency of the ribosomes from 48-h cells in translation, salt-washed ribosomes from 12- and 48-h cells were examined for their abilities to catalyze polyphenylalanine synthesis. Even after salt washing, ribosomes from 12-h cells were about five times higher in specific activity (counts per minute of polyphenylalanine synthesized per absorbance at 260 nm of ribosomes) than equivalent amounts of ribosomes from 48-h cells. Analysis of the proteins of salt-washed ribosomes of the two cell types by acrylamide gel electrophoresis suggests that the relative amounts of individual proteins present on ribosomes from 12-h cells are different from the amounts present on ribosomes from 48-h cells. These results are discussed in terms of the regulation of translation in S. antibioticus.

Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy.

The interphase nucleus of the normal rat hepatocyte has been studied in ultrathin frozen sections after glutaraldehyde fixation and the modification of various staining procedures known to be specific for DNA structures (Moyne's thallium stain, Gautier's osmium-ammine) or preferential for RNP carriers and basic proteins (regressive stains based on the use of EDTA or citrate, negatively charged colloidal iron). The results are comparable to those obtained after classical dehydration and embedding. Particular attention has been paid to the nucleolus and extranucleolar RNP components, such as perichromatin fibrils and granules, as well as interchromatin granules. A striking observation was the uneven size and the strongly increased number of perichromatin granules, and the appearance of a contiguous interchromatin net, containing nucleoproteins. Cryoultramicrotomy without embedding appears to be very useful for the exploration of the nucleus in thick sections which remain sufficiently transparent even with the usual accelerating voltages.

Applying concepts of visual perception to formats of hospital menus.

Standardized printed menu formats for all diets utilizing concepts of visual perception were evaluated in a machine-paced hospital tray-assembly process. Formats of existing menus differed among the various diets. On the redesigned menus, all menu items were arranged in basic groups which were assigned specific positions; groups were accentuated by white strips across the various color-coded selective menus; and accessory items were placed in specific, standard positions on all menus. Criteria for evaluating the effect of using the redesigned menu in tray assembly operations were: overall productivity, individual productivity, and error rate per tray. Data were charted (a) during a control period when the existing menu formats were used to provide baseline data and (b) during an experimental period when the redesigned menu formats were used. Overall productivity was measured by man-minutes per tray. Video tapes of five station operators servicing selected trays were made to study individual productivity. Station operator and checker accuracy were measured in terms of ratio of error-free trays, errors per tray, and errors to possibility of errors per tray. Man-minutes per tray decreased significantly in the experimental period from 2.44 to 2.17--a productivity increase of 11.1 per cent. The individual productivity analysis revealed no significant changes from control to experimental periods. Accuracy of the tray assembly station operators improved significantly. Decreases in ratio of mean number of errors to possibility of errors per tray were recorded in the experimental period. The error rate per tray decreased 44.9 per cent from 0.48 to 0.26, and the ratio of errors to possibility of errors per tray decreased from 6.3 to 3.5 per cent. The percentage of error-free trays rose from 69.9 to 80.9 per cent. Checkers' errors per tray did not change significantly from control to experimental period when data for the two periods were compared. This study provides a practical means of increasing productivity and improving accuracy of the machine-paced tray assembly process.

The antibody responses to myelin basic protein (BP) in Lewis rats: the effects of Bordetella pertussis.

A time-course study was made of the systemic humoral immune response of Lewis rats to myelin basic protein (BP) as influenced by the dosage of ancillary pertussis adjuvant. Peak activities were observed 5 to 7 weeks after injection. When injected proximal to BP and Mycobacterium butyricum in complete Freund's adjuvant (CFA), Bordetella pertussis at the level of 4 billion organisms doubled the antibody-binding activity of rat sera for 125I-labeled BP as compared to activities obtained with 0, 2, 6, or 8 billion. The severity of clinical symptoms of experimental allergic encephalomyelitis (EAE) at the end of the 2nd week was greatest in rats receiving 64 billion organisms, the very same rats that displayed a severely dampened humoral immune response to BP 5 weeks later. When pertussis was injected i.p. rather than proximal to the CFA mixture, the time-course of the humoral immune response displayed a different profile--unusually high binding activities at the time of onset of EAE that fluctuated back and forth from high to low and that eventually dampened to an intermediate level.

Antigenic analysis of Chlamydiae by two-dimensional immunoelectrophoresis. II. A trachoma-LGV-specific antigen.

Two-dimensional immunoelectrophoresis was utilized to study precipitins in hyperimmune rabbit serum made against chlamydiae and from patients with chlamydial infections. An antigen of Triton X-100-solubilized L2/434/Bu organisms with an electrophoretic mobility of 0.65 relative to bovine serum albumin at pH 8.6 was excised from the agarose gel of electrophorograms as antigen-antibody complexes and used to immunize rabbits. A monospecific antiserum to antigen 0.65 was obtained that reacted with Trachoma-LGV strains L2/434/Bu, B/TW-5/OT, and K/UW-31/Cx, but not with the mouse pneumonitis (Nigg) strain or the psittacosis strain meningopneumonitis (Cal-10). The Trachoma-LGV specificity of antigen 0.65 was further shown by indirect immunofluorescence straining with the monospecific antiserum of chlamydial inclusions in infected HeLa cells. Precipitins with a specificity for antigen 0.65 were indentified in 15 of 18 sera from patients with diagnosed Chlamydia trachomatis infections LGV, trachoma, nongonococcal urethritis, and nongonococcal cervicitis by using monospecific antiserum to antigen 0.65 in the peak suppression test. Thus, antigen 0.65 appears to be a Trachoma-LGV-specific antigen that has considerable promise for serodiagnosis.

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.

Inverse relationship between net electric charge on the antigen and that on the sensitized cell in cellular immune response: demonstration with basic encephalitogen of the brain.

An inverse relationship exists between the net-electrical charge of immunogens and the antibodies elicited (1). The cellular basis of the net charge phenomenon has been established for both positively and negatively charged immunogens, by cell separation techniques over columns of opposite charge (7, 8). To establish whether this phenomenon can be extended to include cell-mediated immunity, the response to basic encephalitogenic protein (BE) which induces experimental allergic encephalomyelitis (EAE) was now investigated. Lymph node cells from sensitized strain 13 guinea pigs were fractionated over positively and negatively charged columns and compared to unfractionated cell populations in two assay systems: (a) in vitro response to BE in terms of lymphocyte transformation and (b) the passive transfer of EAE to unsensitized syngeneic recipients. The response was found to be confined to the fraction of cells eluted from glass bead columns, namely, the more negative cells. Cells eluted from poly-L-lysine-coated glass bead columns (i.e., positive cells) were devoid of the capacity to respond to this antigen either in vivo or in vitro. It was previously established that thymocytes rather than bone marrow cells account for the inverse charge phenomenon as assayed by T-helper-cell function in in vivo antibody production (8). We have now extended the inverse charge effect to include cell-mediated immune response of the delayed hypersensitivity type.

[Representative enzymes of energy supplying metabolism in the normal and denervated human brachial biceps, deltoid and anterior tibial muscles (author's transl)].

Representative enzyme activities of energy supplying metabolism were measured in muscle specimens of brachial biceps, deltoid or anterior tibial muscle of patients with affections of the peripheral nerves. Simultaneously performed measurements of the same enzyme activities in the contralateral normal muscles served as a control. 5 patients suffered from a lesion of the brachial plexus, 7 patients had a paralysis of the axillary nerve, and 8 patients had a peroneal paralysis. In all denervated muscles no electrophysiological signs of reinnervation were present. The activities of glycogen phosphorylase, triosephosphate dehydrogenase, lactate dehydrogenase and alpha-glycerophosphate dehydrogenase were found to be highest in the normal brachial biceps muscle. Lower activities were measured in the normal deltoid and anterior tibial muscle. The oxidative enzymes, 3-hydroxyacyl-CoA dehydrogenase and citrate synthase as well as hexokinase, showed no significant difference from the levels of the control. It is suggested that a probable factor determining the differences of the enzyme activities of glycogenolysis, glycolysis and alpha-glycerophosphate oxidation between brachial biceps, deltoid and anterior tibial muscle, might be the pattern of impulse activity in the motor nerves of these muscles. The enzyme activities of glycogen phosphorylase, triosephosphate dehydrogenase, lactate dehydrogenase and alpha-glycerophosphate dehydrogenase, decreased rapidly during the first 2 months after denervation in the brachial biceps, deltoid and anterior tibial muscle, whereas the decrease was slight during the following months. The activities of the oxidative enzymes (3-hydroxyacyl-CoA dehydrogenase and citrate synthase) showed no significant change after denervation. The metabolic difference of glycogenolysis, glycolysis and alpha-glycerophosphate oxidation between the three muscles was no longer maintained. The possible causes of the deeply decreased enzyme activities of glycogenolysis, glycolysis and alpha-glycerophosphate oxidation, as well as the causes of the unchanged oxidative enzyme activities and of the increased hexokinase activity after denervation in the human brachial biceps, deltoid and anterior tibial muscle, are discussed.

Histochemistry of the keratohyalin granules in human oral leukoplakia.

The keratohyalin granules from 25 human oral leukoplakias, showing benign hyperorthokeratosis histologically, were examined employing a series of histochemical techniques. The tissues were fixed in 10% neutral buffered formalin, 80% methanol, or Carnoy's fluid. The keratohyalin granules stained intensely with Pauly's reagent, Congo red and Harris hematoxylin, indicating the presence of proteins. This was confirmed by abolishing the staining reaction by pretreatment with proteolytic enzymes. The keratohyalin granules also reacted with methyl green-pyronin by staining pink at their peripheries; this staining was abolished by pretreatment with ribonuclease, indicating the presence of ribonucleotides. The keratohyalin granules partially stained with toluidine blue and colloidal iron, indicating the presence of acid polysaccharides. The keratohyalin granules did not react with the Feulgen reagent, suggesting the absence of DNA. Our studies indicate that the keratohyalin granules in human oral leukoplakia are primarily protein(s) complexed with polyribonucleotides. The presence of a carbohydrate moiety suggests the possibility of a protein-polysaccharide component in the granules.

Regulatory effects of steroids on the pituitary response to LH-RH.

The effects of continuous intravenous administration of luteinizing hormone-release hormone in conjunction with physiological or pharmacolog ical doses of estradiol (E2), progesterone (P), 17alphaOH-progesterone (17-P), 20alphaOH-progesterone (20-P), chlormadinone acetate (CA), testosterone (T) and 5alpha-dihydrotestosterone (5-DHT) on pituitary response was evaluated in normal and postmenopausal women, normal men, and 1 patient with Klinefelter's syndrome. An all subjects, except the Klinefelter's syndrome patient, 132 mcg/hour/6 hours of E2 blocked the pituitary response to LH-RH, although a dose of 6.6 mcg/hour/6 hours was not markedly effective. None of the progestational steroids blocked the pituitary response to LH-RH. However, the dual administration of CA with E2 antagonized the pituitary response in normal and postmenopausal women. 600 mcg/hour/6 hours of T partially inhibited the pituitary resp onse in men, but had little effect on postmenopausal women and the Klinefelter's syndrome patient. The pituitary response to LH-RH was not inhibited by 5-DHT. The results indicate that sex steroids can modulate the hypothalamic-pituitary axis, though estradiol by itself does not appear to increase the pituitary response to LH-RH since it is not solely responsible for the pituitary responsiveness to LH-RH at the mid-luteal phase.

Relevance of "significant bacteriuria" to aetiology and diagnosis of urinary-tract infection.

28 of 74 female domiciliary patients who presented with urinary-tract symptoms did not fulfil the usually accepted laboratory criterion required for confirmation of urinary-tract infection (i.e., a urinary bacterial viable count in excess of 100 000/ml). The progress of the disease was similar in non-bacteriuric and bacteriuric patients. In addition, a significant proportion of the bacteriuric group did not show the response to therapy expected from the results of in-vitro tests of antibiotic sensitivity and urinary antibacterial activity. To explain these findings, it is postulated that bacteria may not be primary pathogens in uncomplicated urinary-tract infection even where "significant bacteriuria" is demonstrated.

Clinical applications of bone-marrow culture.

The use of in-vitro culture methods for studying human haemopoietic cells has advanced greatly since 1970. These methods have contributed to our understanding of the mechanisms controlling granulopoiesis though the physiological role of colony-stimulating factor needs further clarification. In leukaemia they offer an approach to the study of possible causal factors and to the characterisation of leukaemic-cell defects. Results already obtained support the concept that the bone-marrow in acute myeloid leukaemia consists of coexisting populations of normal and leukaemic cells, with a leukaemic clone predominating in relapse and normal clones regenerating in remission. For the individual patient, in-vitro methods may prove useful in assessing prognosis and in confirming the completeness of remission; the detection of early relapse may then indicate the need for changing or re-instituting therapy. Further studies may aid the classification of the "preleukaemic" states and may help in the identification of the various causes of neutropenia.

[Histochemical study of the effect of phosphoroorganic esters Z-50 (fenchlorfos) and Z-51 (bromophos) on cholinesteras activity in the course of the muscle phase in experimental trichinellosis in mice].

The studies were carried out on mice between the 14th and 19th days, i.e., in the period of maximum penetration of T. spiralis larvae in the host's muscles. Phosphoro-organic esters were given orally to experimental animals in oil solution, at the following doses: Z-50 -- 150 mg per kg of body weight and Z-51 -- 100 mg per kg of body weight. The influence of esters on the activity of cholinesterases was investigated with the Koelle-Friedenwald method, modified by Gomori. The aim of the study was to establish the joint activity of the migrating Trichinella larvae and phosphoro-organic esters on the organism of the host. Z-50 and Z-51 penetrate in therapeutic doses the striated muscles rather weakly. 24 hrs after these compounds were given, acetylcholinesterase (AChE) activity was inhibited in motor end-plates in about 30%, and in muscle fibres infected with T. spiralis larvae in about 60-70%. Pseudocholinesterase (PChE) activity was stronger inhibited than AChE. 24 hrs after applying Z-50 and Z-51 PChE was inhibited in about 90%. Of the two phosphoro-organic esters being examined Z-51 stronger inhibited the cholinesterases activity than Z-50. It was found that the efficiency of phosphoro-organic esters in the course of trichinellosis depends on its ability of infiltration into the host's muscles and on the degree of inhibition of the active cholinesterases in the motor end-plates. Attention was drawn to the fact that increased activity of cholinergic system is one of the main factors in the patholgenesis of the second phase of trichinellosis, i.e., the migration and penetration of the larvae in the muscular fibres of the host.

Oncornavirus-like particles from cultured bone marrow cells preceding leukemia and malignant histiocytosis.

Particles with the density and enzymatic activity characteristic of known oncornavirus have been previously described in bone marrow cells from patients with leukemia in relapse and in remission. We have confirmed these findings and studied two patients in whom preleukemia was among the diagnostic considerations. Following cultivation of bone marrow from these patients for 1 week in conditioned media with dexamethasone, a high-speed pellet of the supernatant fluid and disrupted cells was prepared and analyzed on a sucrose gradient for enzymatic activity characteristic of RNA-directed DNA polymerase (reverse transcriptase). Peaks of endogenous DNA polymerase activity showing ribonuclease sensitivity and/or stimulation with the synthetic template poly(rC)-(dG)12-18 were demonstrated in both patients at densities of 1.15 to 1.19 and 1.21 to 1.24 g/ml. Subsequently, diagnosis 2 and 4 months after initial evaluation revealed acute myelogenous leukemia and malignant histiocytosis, respectively. Prior studies have suggested a possible etiological significance of such particles in human leukemia. The demonstration of similar particles preceding clinically overt disease in these patients supports this hypothesis and offers the possibility of early diagnosis and treatment.

Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. IV. Isolation of two groups of naturally occurring, idiotypic molecules with specific antigen-binding activity in the serum and urine of normal rats.

In the rat the major histocompatibility locus antigens are determined by the Ag-B locus. In the present article evidence is presented that the sera and urine of normal adult rats contain naturally occurring antibody-like molcules with reactivity to allogeneic Ag-B antigens. Such molecules can be shown to contain both antigen-binding capacity for the relevant antigens and the idiotypic markers signifying such specific reactivity. The molecules could be shown to be composed of two groups of molecules, one around 7S IgG in size and the other around 35,000 in molecular weight. Only the smaller molecules were found in the urine. From other data we know that the 7S molecules are produced by B lymphocytes and the 35,000-molecular-weight molecules by T cells. Purified natural anti-Ag-B factors, when inoculated into rabbits or chickens, lead to the production of specific anti-idiotypic antibodies that will selectively inactivate rat T lymphocytes with the capacity to react against the relevant Ag-B antigens while leaving other reactivity intact. We thus conclude that the present system allows the purification of naturally occurring idiotypic B- and T-cell products with antigen-binding specificity for further biochemical and functional analysis.

Mediastinoscopy and bronchial carcinoma: experience with 600 mediastinoscopies.

Of 600 mediastinoscopies carried out from 1966 to 1973, 479 were performed to assess the operability of a pulmonary carcinoma. Of these, 206 (43%) were positive and 273 (57%) were negative. Of the 161 patients found positive during an initial period, 147 were refused operation; the remaining 14 were considered suitable candidates for operation, either because only one homolateral lymph node site was involved or because there was a concomitant osteoarthropathy. The tumour was irresectable in one of these 14 patients who died after 3-5 months; curative resection was possible in one and palliative resection in 12 patients. These 12 patients all died within a year. Of the 184 patients found negative during an initial period, 149 were treated by operation. The tumour proved irresectable in seven (5%), while curative resection was possible in 113 (76%) and palliative resection in 29 (19%) patients. Comparison with the period 1957-63, when in the same hospital resection was performed after a negative Daniels' (scalene node) biopsy, shows that the tumour was irresectable in 25 (20%) of the 124 patients with a negative biopsy, while curative resection was possible in 43 (35%) and palliative resection in 56 (45%) patients. During a second period, patients with a positive mediastinoscopy were in principle refused operation. Of 89 negative patients, 81 were treated by operation. No tumour was found to be irresectable; curative resection was possible in 63 (78%) and palliative resection in 18 (22%) patients. An operation for bronchial carcinoma was performed on 167 patients between September 1970 and September 1973 after a negative mediastinoscopy in 95, and without mediastinoscopy in 71 patients, either because of a peripheral tumour (70) or because of a tumour relapse after two years (1). The resection was palliative in 11% of the 71 cases, but in only one patient with a peripheral tumour could a mediastinoscopy have been positive. Finally, an operation was performed on one patient with a positive mediastinoscopy and a tumour relapse after six years. A survival study was made of the first 100 patients with pulmonary carcinoma, operated on between September 1970 and March 1972 and with a follow-up from a minumum of two years to a maximum of 3-5 years. The early mortality averaged 10% and was higher after pneumonectomy than after lobectomy. The late mortality was 16% after curative lobectomy, 38% after curative pneumonectomy, and 83% after palliative pneumonectomy. The survival after 2 to 3-5 years was 63%.

Immunological activity of the peptidoglycan.

Studies on the immunology of peptidoglycan received impetus from the initial observation that the rabbit Group A-variant streptococcal antisera were a rich source of antibodies to peptidoglycan. Indeed, quantitative precipitin studies revealed concentrations as high as 7-10 mg/ml of antiserum. The amount of antibody after Group A-variant streptococcal immunization is much greater than the amount in the sera following immunization of rabbits with the Group A or C streptococci. Furthermore, earlier studies had shown that the purified peptidoglycan obtained as a residue following extraction of streptococcal cell walls with hot formamide was a poor antigen. Both the hexosamine polymer and the peptide moiety are antigenic. Use of the solid phase synthesized pentapeptide L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala and related similar peptides facilitated the determination of the fine structure of the immunodominant site of pentapeptide. The evidence points to the C-terminal D-Ala-D-Ala as the immunodominant determinant. Because of the similarity of the peptidoglycans of a number of different bacteria, it would be anticipated that they would cross-react immunologically, and this has been shown to be the case. The biological and medical significance of antibodies to peptidoglycan has yet to be determined. Certainly the exposure to this ubiquitous substance which occurs in all the indigenous bacteria of the respiratory and the gastrointestinal tract must mean that from an early age and through advancing years there is a constant stimulation of the immune response to peptidoglycan. Because the immunochemistry of these substances is now firmly established, there is a scientific basis for proceeding with the medical and biological implications of peptidoglycan immunity.

Liver biopsy in methotrexate-treated psoriatics-a re-evalution.

Two-hundred and eighty-six liver biopsies were performed in 139 psoriatics on treatment or considered for treatment with methotrexate. In 56 psoriatics included in this study both pre- and post-methotrexate biopsies were performed, the average methotrexate dose being 936 mg. None of the data showed statistically significant differences between pre- and post-methotrexate biopsies, with the exception of an increase in fattly infiltration, found when comparing all pre-methotrexate biopsies with the total number of latest post-methotrexate samples. As expected, alcohol seemed to be significantly associated with liver fibrosis in pre-methotrexate biopsies. An patients, although potassium arsenite alone has not been proved to be the cause of liver damage among psoriatics included in this study. While only 1 of 22 psoriatics with a total normal biopsy had been on arsenite, 6 of 18 of the same group of psoriatics who had fibrosis had been on this drug earlier. Although no statistically significant differences related to fibrosis and cirrhosis could that in three cases liver cirrhosis did appear in a biopsy from a methotrexate-treated psoriatic who had signs of fibrosis of cirrhosis in a pre-methotrexate biopsy. This incidence is low in relation to the total number of patients treated. The relatively low incidence of cirrhosis found in the present study, as in earlier studies by our group is believed to be due to the use of an intermittent dosage schedule. The study showed that early fibrosis and cirrhosis seem to appear, with very minor abnormalities in laboratory results. This finding indicates the necessity of performing liver biopsies in the control of psoriatics on long-term methotrexate therapy. The difference between biopsies from psoriatics and liver biopsies from control patients may indicate that severity of disease may be a complicating factor in the pathogenesis of the liver damage.

[On the possibility to demonstrate the presence of catecholamines in enterochromaffin cells: critical review (author's transl)].

Various data contributing to support, after Lillie et al., the presence of catecholamines instead of (or in addition to) 5-hydroxytryptamine in the enterochromaffin cells, are submitted to analytical criticism by the Author. Topics of the discussion are mainly the behaviour of the diazo-reaction (in acid medium), the fluorescence induced by formaldehyde, and various reactions carried out either directly or following a chemical block. The Author demonstrates that 5-HT is present in the enterochromaffin cells and that it is impossible, at present time, to prove the presence of catecholamines in these cells on the basis of histochemical data. The Author debates also how biochemical or pharmacological tests may supply the lacking of histochemical proofs in demonstrating the presence of catecholamines; it is possible by applying these procedures to study situations like those postulated by Lillie et al., extending a comparative research also to other elements of the enterochromaffin cell system after ERSPAMER; the examples here reported however do not agree perfectly in some of the cases with these views.

The effect of Trimepranol on thrombocyte function and histamin release in the rat.

Trimepranol a beta adrenergic blocking drug released serotonin from rat isolated and plasma-rich thrombocytes in vitro. The release was time- and dose-dependent. With the same concentrations this drug inhibited the histamine release from isolated rat mast cells. Aggregation of isolated or plasma-rich thrombocytes induced by ADP was inhibited by Trimepranol. The inhibition was dose, time dependent and reversed by calcium ions.

Immunology of DNA. III. Crithidia luciliae, a simple substrate for the determination of anti-dsDNA with the immunofluorescence technique.

C. luciliae are hemoflagellates nonpathogenic for man and easy to culture. They have a giant mitochondrion, in which the mitochondrial DNA is concentrated in a single large network, the kinetoplast. When used as a substrate for the indirect immunofluorescence technique, studying sera from patients with SLE, we could demonstrate a very good correlation between this test and the Farr assay for the demonstration of antibodies to double-stranded DNA. Although the sensitivity of both techniques is on the same order of magnitude, the IF technique has the following advantages over the Farr assay. It is easy to perform in laboratories equipped for autoimmune serology. It possesses an intrinsic check on the immunoglobulin character of the DNA-binding activity. It allows one to determine the Ig classes and subclasses of antibodies to DNA. It permits study of complement fixation to antibodies without interference of Clq fixation to DNA or anticomplementarity of the serum. There is an absence of interference with antibodies to single-stranded DNA.

Two IgG1 (IgF) subclasses in mice: presence in normal serum and representation within lymphocyte and plasma cell populations.

Monospecific anti-gamma1 sera were prepared in rabbits against each of two IgG1 mouse myeloma globulins isolated from myeloma A2 and MOPC21. Both these sera react with IgG1 of normal mouse serum by immunoelectrophoresis and cannot be distinguished any further. The antibodies contained in the sera were cross-isolated on immunoadsorbents containing either the A2 or MOPC21 antigenic material and tested by immunoelectrophoresis against normal mouse serum. An interesting spur was formed between these two antibodies. The two isotypes thus revealed in normal serum are tentatively designated IgG1a (globulin A2) and Ig G1b (globulin MOPC21). With live peripheral lymph node cells almost all lymphocytes which stain with antikappa antibodies (B lymphocytes) also stain with the specific anti-gamma1a antibodies, but only 5-10% stain with the specific anti-gamma1b antibodies. On fixed spleen cells (C57B16 mice) the anti-gamma1a antibodies stain almost identical percentages of plasma cells as anti-kappa antibodies: 1.8% are gamma1 and 2.2% are kappa. Anti-gamma1b antibodies stain only 5% of kappa-positive plasma cells. The % values estimated on both the normal lymphocyte and plasma cell populations were not significantly affected by inhibtion experiments of the specific anti-gamma1 antibodies performed with isolated myeloma globulins representative of several Ig classes.

Further studies on the specificity of the N- and C-terminal antigenic determinant of hen egg-white lysozyme.

The specificity of the N- and C-terminal antigenic determinant (P17: sequence Lys1-Cys6-Asn27, Trp123-Cys127-Leu129) of hen egg-white lysozyme (HL) was studied in more detail. In a Scatchard plot of the binding of 14C-acetyl HL with guinea pig purified anti-P17 antibody experimental values bent sharply near r=1. This suggests the presence of two antibody populations with different affinities for HL or possible steric hindrance in the binding of a second HL molecule to the second binding site of the antibody molecule. The antigenic activities of various peptides were tested by measuring their inhibition of the binding of 14C-acetyl-P17 with the anti-P17 antibody. Only P17 and P17t (sequence Lys1-Cys6-Homoser12, Trp123-Cys127-Leu129) were inhibitory, with KI values of 2.0 times 10(4) and 8.1 times 10(3), respectively. These results indicate that the direct binding site of P17 to anti-P17 antibody may be located in the terminal portion of P17 (sequence Lys1-Cys6-Homoser12, Trp123-Cys127-Leu129) while the rest of P17 may be important in maintaining the conformation of this determinant. The single disulphide blood involved in this determinant is essential for manifestation of immunological activity.

Detection of structural differences between nuclear and mitochondrial glutamate dehydrogenases by the use of immunoadsorbents.

Structural differences between crystalline mitochondrial and nuclear glutamate dehydrogenases from ox liver have been detected by immunological techniques. Antisera prepared against each enzyme precipitate both glutamate dehydrogenases; upon immunodiffusion, the antiserum against the nuclear enzyme gives a line of incomplete identity with the two antigens, whereas the antiserum against the mitochondrial enzyme gives a line of complete identity. Fractionation of the antibodies contained in each antiserum by means of an immunoadsorbent, to which the nuclear or the mitochondrial enzyme has been covalently linked, shows that nuclear glutamate dehydrogenase (GDH) contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, whereas the latter appears to have no antigenic portions which are not present in the nuclear antigen, in accord with the results of immunodiffusion. The antibodies against determinants common to both enzymes precipitate and inhibit them, whereas the specific anti-nuclear GDH antibodies precipitate but do not inhibit the nuclear antigen.

Palliation in the management of laryngeal cancer. Surgical concepts in palliation of advanced disease.

Every science has limits to its operation, including medical science concerning malignancy. Beyond a certain stage of a disease, palliation is the only recourse. The word "palliation" amounts to an acceptance of defeat by a clinician while trying to salvage his cancer patients. It means that the patient should breathe and eat without much pain till death takes pity on him. Palliative surgery in laryngeal cancer amounts to doing tracheostomies and gastrostomies and administering painkillers. Most of my cases belong to this category. I extended the accepted parameters of surgical excisions for primary lesion and metastatic nodes. These excisions include laryngectomy with cervical esophagectomy, total laryngectomy, total cervical esophagetomy, total glossectomy, and total mandibulectomy. The extended radical neck dissections include carotid artery, vagus nerve, and sympathetic trunk on one side. Removal of these so-called vital structures was not only compatible with life but proved curative in 20 per cent of these cases.

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat. III. Paraventricular and supraoptic neurons during lactation and dehydration.

The ultrastructural features of paraventricular (PVN) and supraoptic (SON) neurons and of their axons were studied in lactating and dehydrated rats. Under both conditions of stimulation, the PVN and SON neurons and their axons enlarge. The protein synthesizing apparatus of the neurons becomes activated, but the number of neurosecretory granules (NSG) is decreased. No differences are seen between the PVN and SON neurons during lactation or dehydration. The similarity and simultaneity of the response of the PVN and SON neurons to these two different stimuli is discussed in the light of the theory of nuclear and neuronal specialization for the production of only one hormone. After prolonged lactation of over 2 1/2 weeks' duration, neurons with extreme vacuolation of the rough endoplasmic reticulum (RER) appear in the PVN and SON; the vacuolated neurons appear earlier and predominantly in the PVN involving a maximum of 10-15% of all PVN neurons. Vacuolated neurons were never seen in either nucleus during dehydration of up to 6 days' duration. The vacuolation is suggested to represent an exhaustion phenomenon due to an intense, long-lasting stimulus for oxytocin synthesis. The predominant location of the vacuolated neurons in the PVN supports the theory that oxytocin is produced predominantly in the PVN. The decrease in the number of NSGs during these states of enhanced hormone secretion is considered to corroborate the proposed existence of an extragranular fast axoplasmic transport mechanism in PVN and SON neurons. The possible existence of a reuptake mechanism into NSGs, similar to that in the vesicles of monoaminergic nerve endings is discussed.

Amniotic fluid alpha2-macroglobulin and the antenatal diagnosis of spina bifida and anencephaly.

Alpha2-macroglobulin (AMG) concentrations have been measured in amniotic fluids from 33 pregnancies where the outcome was an infant with a neural tube defect. AMG ranged from 1.3 to 50 mug/ml in these samples, but was undetectable (is less than 1 mug/ml) in matched controls. Since the abnormal samples included four cases of spina bifida and eight cases of anencephaly before 22 weeks of pregnancy, measurement of AMG concentrations may be useful in the early antenatal diagnosis of neural tube defects. In some cases it gave clearer results than those obtained by measurement of amniotic fluid alphafetoprotein. However, care must be excercised to ensure that amniotic fluids are not contaminated by blood.

Electroencephalographic correlates of myoclonus.

In order to overcome various drawbacks of the conventional polygraphic study of a relationship between myoclonus and EEG, the EEG preceding and following the myoclonic jerk was simultaneously averaged by the CNV program. The subjects were 7 patients presenting with myoclonus of various kinds. The conventional polygraphs showed various paroxysmal EEG activities in 4 patients, but none of those paroxysmal activities was temporally related to myoclonus except for one case. As a result of the present averaging technique, 2 patients with cerebellar ataxia with intention myoclonus showed myoclonus-related EEG spikes or spike-and-slow-waves in the contralateral central or centroparietal region. These myoclonus-related spikes preceded the myoclonus by 10-17 msec, suggesting the presence of a discharging focus in the deep cerebral structures, rather than in the cerebral cortex, in these cases. Two other patients, one with resting myoclonus and the other with postural myoclonus, showed myoclonus-related slow waves on the contralateral hemisphere. This previously undescribed method of averaged polygraphic recording will be very useful in detecting an EEG correlate of spontaneously occurring myoclonus.

Time trends and periodic cycles in REM sleep eye movements.

Eye movements during REM sleep episodes were tabulated in 16 young adults. REM episodes were then broken down into four ranges according to length in min: (1) 11.0-21.3; (2) 21.7-29.7; (3) 30.0-42.3; (4) 42.7 or longer. These data were then analyzed for linear and quadratic trends. Eight episodes had a significant linear trend, 10 had a significant quadratic trend, 7 had both linear and quadratic trends, while 12 had no trend. The residuals from the best-fitting polynomial curve were then subject to a spectral analysis. In addition, 2 long periods of pre-sleep wakefulness (approximately 2 h each) were also analyzed. In general, the spectral analysis revealed the dominant presence of a slow cycle (period of 10 min to about 30 min) the exact period of which varied according to the length of the REM episode. A binomial probability test indicated that the presence of slow cycles was significant in REM episodes except for those in the 21-30 min range. For the episodes of wakefulness, a dominant slow cycle was found in both cases. The results give the impression of similarity in the periodic organization of eye movements during REM sleep and waking. The data also indicated that an ultradian (70-150 min) cycle was present in eye movements during sleep and waking. Further, the finding of a decrease in eye movements before sleep onset, coupled with previous reports of an increase in eye movement after sleep onset, indicate the presence of a circadian cycle.

[The use of the cell separator in the treatment of leukemia].

In order to provide leukemic patients during the critical granulocytopenic stage with a sufficient amount of granulocytes a blood cell separator with a continuous extracorporeal circulation was developed. This permits to obtain up to 3.0-10(10) leukocytes during a 4---5 hours period from a single donor. According to our own experiences with 20 leukophereses performed in 13 healthy donors by the use of the AMINCO cell separator an average of 1.17-10(10) leukocytes with a granulocytic portion of 61% was collected per run. In two cases of agranulocytosis and septic fever (one case of pseudomonas septicaemia) the repeated administration of leukocyte concentrates, while specific antibiotic therapy was continued, led to a marked improvement over a longer period of time. Furthermore thrombocyte concentrates up to 7.0-10(10) platelets can be obtained by the cell separator. Applied as depletory method in the treatment of CML and CLL leukopheresis may rapidly diminish the peripheral leucocyte count while spleen and lymphomas decrease in size at the same time. A 20% reduction in cell count may be achieved by a serie of 3---4 leukophereses. Also the use of the cell separator in the treatment of makroglobulinemia by plasmapheresis is discussed.

Intermittent hyperthyreosis -- a heat stress syndrome.

Intermittent hyperthyreosis occurs under various forms of stress, especially heat stress. The clinician may diagnose such cases as masked or apathetic hyperthyroidism or "forme fruste" hyperthyreosis or thyroid autonomy. As most routine and standard tests may here yield inconsistent results, it is the patients' anamnesis which may provide the clue. Our Bioclimatology Unit has now seen over 100 cases in which thyroid hypersensitivity towards heat was the most prominent syndrome: 10-15% of weather-sensitive patients are affected. The patients complain before or during heat spells of such contradictory symptoms as insomnia, irritability, tension, tachycardia, palpitations, precordial pain, dyspnoe, flushes with sweating or chills, tremor, abdominal pain or diarrhea, polyuria or pollakisuria, weight loss in spite of ravenous appetite, fatigue, exhaustion, depression, adynamia, lack of concentration and confusion. Determination of urinary neurohormones allows a differential diagnosis, intermittent hyperthyreosis being characterized by three cardinal symptoms: 1. tachycardia -- every case with more than 80 pulse beats being suspect (not specific); 2. urinary histamine -- every case excreting more than 90 mug/day being suspect. Again the drawback of this test is its lack of specificity, as histamine may also be increased in cases of allergy and spondylitis; 3. urinary thyroxine -- every case excreting more than 20 mug/day T-4 being suspect. This is the only specific test. Therapy should make use of lithium carbonate and beta-blockers. Propyl thiouracil is rarely required.

Influence of molecular structure of the tolerogenicity of bacterial dextrans. III. Dissociation between tolerance and immunity to the alpha1--6- and alpha1--3-linked epitopes of dextran B1355.

Dextran B1355 induces a direct PFC response detectable with dextran B512-sensitized red cells which is directed towards an alpha1--6-linked glucose epitope. This response is distinguishable from the alpha1--3-linked specificity assayed by homologous sensitization in that: (a) it is totally suppressed in donors previously rendered tolerant of B512; (b) the PFC are sensitive to inhibition by B512 and isomaltohexaose. The alpha1--6 epitope of B1355 is less immunogenic in BALB/c mice than that with alpha1--3 linkage, inducing a lower amplitude of response and requiring a 100-fold greater minimal dose, while conversely, it is the more effective tolerogen. No alpha1--6-specific response develops in 50 per cent of mice given 10 mg of B1355 and all become totally unresponsive within 14 days. This tolerant state remains stable when spleen cells are transferred to irradiated recipients. By comparison, parallel depression of the alpha1--3 response is not great and rapidly lost by similar transfer. No correlation was observed between the levels of alpha1--6 suppression and alpha1--3 response induced by 10 mg of B1355 in individual mice. The dissociative aspects of the responses to these two epitopes present on the same molecule are discussed in relation to some current theories of B-cell tolerance induction. It is argued that the present findings are contrary to those models which attribute a causal role to mitogenic overstimulation or failure to generate an extrinsic 'second signal'.

Cell co-operation and hapten--carrier complexes.

The co-operation of spleen cells of carrier- and hapten--carrier-primed mice in antibody formation against the hapten part of complexes was studied in 550 rad whole body irradiated mice. Hapten--carrier complexes were prepared with the 2,4-dinitrophenyl group (DNP) as a hapten and heterologous bovine serum albumin (BSA) and isologous mouse immunoglobulin (MIg) as carriers. Priming of donor mice with carrier alone did not prepare for a secondary (IgG) response in the recipients of hapten--carrier. Priming of donors and challenge of recipients with the same hapten--carrier complex resulted in high IgG responses. Whereas donor and recipient immunization with complexes differing in the carrier did not give a secondary response, addition of cells of donors immunized with the carrier of the complex used for challenge, resulted in a secondary response. This was only possible when at least one of the complexes had an intermediate hapten:carrier ratio. Only an IgM or a low IgG response was obtained if both complexes had a high hapten:carrier ratio. Three determinants, namely hapten and carrier groups and new antigenic determinant (NAD), are suggested for antibody formation against hapten--protein complexes. In vivo treatment of donor cells with anti-thymocyte serum (ATS) or anti-plasma cell serum (APCS) and complement (C) suggested that: (1) T-cell epitopes are present on the carrier; (2) DNP groups are B-cell epitopes; (3) NAD and possibly DNP are T-cell epitopes; (4) synergism exists in the collaborative antibody response of B cells recongnizing DNP, T cell recognizing carrier and T cells recognizing NAD. Mitomycin treatment of donor cells was used to test whether cell division was mandatory. While the B cells were sensitive to mitomycin treatment, no effect of this drug was found on the helper activity of T cells.

The histochemical demonstration of aminopeptidase with bromoindolyl leucinamide.

The indigogenic method for aminopeptidase of Pearson et al. (1963) was critically evaluated. The localization obtained with it is not correct due to diffusion artifacts. Ferricyanide cannot be used as an oxidation agent. Based on experiments with other oxidation agents (phenazonium methosulfate, nitro BT, tetranitro BT) a new method was devised. The recommended incubation medium contains 0.9 mM L-N-(5-bromoindol-3-yl) leucinamide hydrobromide (chloride), 0.73 mM tetranitro BT, 0.27 mM phenazonium methosulfate and 0.67 M phosphate buffer pH 7.4. The enzyme activity is indicated by the deposition of tetranitro BT formazan. Results with this method in rat kidney, jejunum, liver, lung, and submaxillary gland, in monkey kidney and jejunum, and in human jejunal biosies are almost identical with those obtained with L-leucyl-4-methoxy-beta-naphthylamide applied in a simultaneous azocoupling procedure. The given principle of the demonstration of aminopeptidase activity with an indolylamine substrate deserves a further exploration in the demonstration of peptidases "in situ" both on optical as well as electronmicroscopical levels.

Distribution of acid phosphatase, beta-glucuronidase, n-acetyl-beta-d-glucosaminidase and beta-galactosidase in cornea of albino rabbit.

Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase.

Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat.

The organization of the brainstem serotonin neuron projection to the hippocampal formation was analyzed in the rat. This projection arises in the raphe nuclei of the midbrain. Following destruction of the midbrain raphe nuclei, chiefly nucleus centralis superior, there is a 72% decrease in hippocampal serotonin content. Injection of tritiated amino acid into the midbrain raphe nuclei results in transport of tritiated protein to the hippocampal formation and this transport is blocked in animals pretreated by intraventricular administration of 5,6-dihydroxytryptamine (5,6-DHT). Autoradiographic analysis indicates that the transport reaches the hippocampal formation primarily via two major pathways, the cingulum and the fornix. Cingulum fibers terminate predominantly in the dorsal hippocampus whereas the fornix distributes throughout the entire hippocampal formation. Some fibers reach the ventral hippocampus from the entorhinal area. Within the hippocampus there is dense labeling in a restricted lamina of the CA1 stratum lacunosum-moleculare with moderate labeling in stratum radiatum. Stratum oriens is sparsely labeled in CA1 and moderately so in CA2 and CA3. Stratum radiatum and stratum lacunosum-moleculare are moderately densely labeled in CA2 and Ca3. The area dentata is sparsely to moderately labeled in the molecular layer and heavily labeled in a thin lamina of the hilar zone immediately beneath the granule cell layer. The remaining hilar zone is moderately labeled. All of the discrete labeling of the hippocampus and area dentata described above is absent in animals pretreated with 5,6-DHT. These observations indicate that serotonin neurons of the midbrain raphe provide a highly organized innervation of the hippocampal formation in the rat.

Determinants of the hierarchy of humoral immune responsiveness during ontogeny.

A model system of ontogeny was utilized to investigate the development of humoral immunity in both AKR and BALB/c mice. Lethally irradiated adult mice were reconstituted with syngeneic fetal or neonatal liver. These mice were immunized at various times after reconstitution with a series of eight antigens: the bacteriophages F2, phiX-174, and T4; the hapten carrier complexes 2,4 dinitrophenyl-bovine serum albumin and fluorescein-bovine serum albumin; and the small proteins: hen egg lysozyme, sperm whale myoglobin, and bovine pancreatic ribonuclease. Subsequent antibody production to the antigens was assayed with either a direct or a modified bacteriophage neutralization technique. Individual mice responded to the various antigens in a sequential pattern which was basically the same for all mice within each strain. However, there was a marked difference between the two strains in the time at which they developed responsiveness to myoglobin. In order to begin to delineate the separate roles played by B and T cells in the generation of this hierarchical response pattern during ontogeny, the development of anti-DNP and anti-FTC activity was examined in carrier-primed mice. Results of this experiment indicated that functional B cell specificities for the two haptens arise at different times during ontogeny. Further studies are needed to determine whether the hierarchical pattern of immune responsiveness observed for the other antigens is a function of sequential appearance of B cell specificities, T cell specificities, or both.

In vitro selection and extended culture of antigen-specific T lymphocytes. II. Mechanisms of selection.

Functional selection of antigen-specific T lymphocytes can be achieved by culturing thymus-dependent (T) lymphocytes from immunized guinea pigs on "monolayers" of antigen-pulsed adherent peritoneal exudate cells (PEC) from nonimmune syngeneic donors. Several aspects of the in vitro selection of T lymphocyte-rich peritoneal exudate lymphocytes (PEL) were studied. It was shown that irradiated adherent PEC were equivalent to nonirradiated adherent PEC in supporting selection cultures, indicating that the lymphocytes harvested at the end of the selection culture derive from the immune donors of the PEL and not from the nonimmune donor of the adherent PEC. The relative importance of specific adherence and specific proliferation for achieving selection was determined by comparing the degree of selection obtained when nonadherent cells were discarded at 24 hr with that noted when the discard step was omitted. It was found that omitting the discard step markedly diminished the degree of selection. On the other hand, blocking proliferation with specific alloantisera after the discard step did not diminish the degree of selection, although it did diminish the cell yield. Thus, specific adherence to antigen-pulsed PEC appeared to be critical in the selection culture procedure. An estimate of the degree of enrichment obtained by the selection culture procedure was obtained by culturing selected cells in an excess of nonprimed PEL, so that auxiliary cells would not be limiting. Under these conditions, it appeared that selected cells were enrichied from 4- to 10-fold in antigen-responsive cells with respect to the initial cell population.

Collaboration of allogeneic T and B lymphocytes in the primary antibody response to sheep erythrocytes in vitro.

This study provides a direct quantitative comparison of the helper effects of allogeneic and syngeneic rat T cells in the production of direct SRBC plaque-forming cell (PFC) responses by B cells in culture. In syngeneic T-B combinations, log-log plots of the number of PFC generated after 5.5 days in culture vs. the number of T cells employed as helpers showed a linear response between 10(4) and 2.5 times 10(5) T cells added. Allogeneic T-B combinations, in which the T cells possess the capacity for reactivity to major alloantigens of the B-cell donor, showed a different dose/response relationship in which PFC responses were decreased at high T/B ratios and augmented at low T/B rations. In this system responses were detected with as few as 10(3) allogeneic T cells. Use of negatively selected allogeneic T populations, specifically depleted of mixed lymphocyte interaction (MLI) and graft-vs-host reactivity for B-cell alloantigens, as helpers gave dose/response curves quantitatively identical to responses with syngeneic T-B combinations and also with F1 T-cell parental B-cell combinations. These data indicate that rat T and B cells need not share a major histocompatibility complex haplotype in order to collaborate effectively in a primary direct PFC response to SRBC in culture. In addition, the PFC response required the combinaed presence of T and B cells as well as antigen in the cultures, a finding consistent with the two signal model of B-cell activation. Finally, the dose/response data obtained suggest the possibility that although SRBC antigen is required in the cultures helper activity with low numbers of normal allogeneic T cells may not depend on T cells having specificity for this antigen.

Axonal transport of RNA during regeneration of the optic nerves of goldfish.

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of 3H-uridine has been studied during various stages of optic nerve regeneration. 3H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Five were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, 14C-proline was also injected into the right eye as a marked of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of 3H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microsocpic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be 3H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of 3H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikaprya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.

[Indications for stereotaxic neurosurgery of patients with multiple sclerosis (author's transl)].

The long-term results of 12 stereotaxic operations on 11 multiple sclerosis patients with incapacitating intention tremor were evaluated and compared with the experiences of other authors. The selection of the patients, the criteria applied for the success and the length of the follow-up period influenced the reported results. Considering not only the relief of the intention tremor but the overall performance after the operation only a certain group of patients seemed to profit by neurosurgical treatment. Applying this criterion of overall performance and evaluation only 3 out of 11 patients in our series had real benefit from the operation. The reasons for this small number of good or moderate results are given with brief discussion of some of the cases. History, course and fatal outcome of one patient are presented in detail together with the neuropathological findings. According to the literature and the limited number of our own cases the following indications for stereotaxic operations on MS patients can be established: 1. Tremor and hyperkinetic movements should be the dominant features of the symptomatology. 2. The overall performance should be essentially improved by the operation. 3. Patients in the terminal stage of the disease gain little from the procedure, whereas patients with longstanding more benign course are the best candidates.

Some experience with 57Co-labeled bleomycin as a tumor-seeking agent.

Bleomycin labeled with 57Co was used as a tumor-localizing agent in 132 patients. In patients with pulmonary tumors the primary localization concentrated radioactivity in 52 of the 54 appropriate cases; out of the 22 clinically known metastases, 19 were visible on the scan; 40 unknown metastases especially in hilus and mediastinum were found by the method and subsequently confirmed. In 22 patients with malignant lymphomas, 18 out of 22 known pathologic lymph glands above the diaphragm were visible on the scan; below the diaphragm the results of scanning in lymph glands and spleen were disappointing, probably because of the disturbing concentration of radioactivity in the kidneys, the bladder, the liver, and sometimes the gut. In 25 patients with various other tumors, 16 out of 22 known localizations above the diaphragm were visible; 2 were uncertain and 4 negative. Below the diaphragm the results were usually negative. In 24 patients with benign lesions, uptake of 57Co-bleomycin was visible on the scintigram in 4 patients with cavitating pulmonary tuberculosis, in 2 with pulmonary infections, in 1 with Caplan lesions of rheumatoid arthritis in the lung, and in 1 with sinusitis ethmoidalis. The significance of these results is discussed.

Autogenous immunity to endogenous RNA tumor virus: reactivity of natural immune sera to antigenic determinants of several biologically distinct murine leukemia viruses.

Sera from normal (C57BL/6XC3H/Anf)F1(B6C3F1) mice reacted with several biologically distinct murine leukemia virus(es) (MuLV) by radioimmune precipitation assays with the use of purified tritiated leucine-labeled virus. The reactivities of this natural antibody to viral envelope antigens of two laboratory strains (Rauscher and Moloney) and two endogenous mouse C-type viruses (AKR and BALB:virus-2) were further analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar patterns of antibody reactivities to AKR MuLV and the two member viruses of the Friend-Moloney-Rauscher group were found. Three major antigenic determinants of the virus envelope, gp71, gp43, and p15, were recognized by and precipitated natural antibody. In all viruses examined, normal B6C3F1 sera precipitated comparable amounts of gp71 and gp43. However, compared with the other viruses, the amount of p15 (relative to the glycoproteins) precipitating from BALB:virus-2 was significantly lower. This appears to be due to a lesser amount of p15 on the xenotropic virus. While heterologous antisera to purified gp71 and p15 of MuLV reacted to a certain degree with rhabdomyosarcoma virus 114 and rat leukemia virus, natural mouse antibody did not. These results suggest that MuLV have common antigenic determinants recognized by natural antibody, and that the reactivities of natural antibody in an autogenous immune response are restrictive in contrast to immune antibody produced in a heterologous host.

Surface structure of Uukuniemi virus.

Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques. The spherical virus particle measures about 95 nm in diameter. Its envelope consists of a 5-nm thick membrane covered by 8- to 10-nm long surface projections. These are composed of two polypeptides species of about the same size. Both of them can be removed by digestion with the proteolytic enzyme thermolysin except for a small fragment. The enzyme-treated particles are smooth surfaced and extremely deformable. The glycopolypeptides are clustered to form hollow cylindrical morphological units, 10 to 12 nm in diameter, with a 5-nm central cavity. Both negative staining and freeze-etching suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice. The membrane to which the surface subunits are attached is probably a lipid bilayer as evidenced by its double-track appearance in thin sections and the tendency of the freeze fracturing to occur within it. The strand-like nucleoprotein appears from thin-sectioning results to be to a large part located in a zone underneath the membrane.

Focal and segmental glomerular hyalinosis and sclerosis in the rat.

A glomerular disease spontaneously developing in Wistar rats was studied by light and electron microscopy and by immunofluorescence techniques. The disease is characterized by the local subendothelial deposition of hyaline material leading to increase of mesangial matrix and the development of adhesions. Immunofluorescence shows deposition of complement and IgM and to a lesser degree also of IgG in these lesions. There is a constant relationship of these early changes with the vascular pole of the glomerulus. It is confirmed that female rats are resistent to the disease as are male rats fed a sodium-deficient diet. A higher protein excretion was found in normally fed male rats as compared to female rats and to rats on a sodium-deficient diet. These differences already existed before the normally fed male rats developed glomerular disease. From these studies it is suggested that an appropriate name for this disease would be focal and segmental glomerular hyalinosis and sclerosis and that hemodynamic factors could be an important etiologic mechanism. The histopathology of the disease bears a striking resemblance to focal sclerosing glomerulopathy with segmental hyalinosis sometimes found in kidneys of patients with an idiopathic nephrotic syndrome.

Cerebral form of high-altitude illness.

Twelve cases of severe altitude illness are reported in which the neurological signs and symptoms dominated the clinical picture. Pulmonary oedema, retinal haemorrhage, thrombophlebitis and pulmonary embolism, bronchopneumonia, and coronary-artery disease were also present in several of the patients but the primary problem seems to have been cerebral oedema. Other published cases support this impression. Patients who were returned to low altitude early in the disease fared well; two patients died, and in both cases evacuation had been delayed. The most effective prevention lies in slow ascent, though in one case reported here the rate of climb was well within the recommended limit. Recommended management is rapid descent to low altitude at earliest indication of cerebral or pulmonary oedema, intravenous dexamethasone or betamethasone in large doses, hydration, diuresis (frusemide has been most used), and perhaps other intravenous therapy with hyperosmolar materials such as mannitol, urea, 50% saline, or 50% sucrose. Prognosis is good if descent and treatment are started early, but permanent damage may be anticipated if the patient is unconscious for any prolonged period before descent.

Prostaglandin, slow-reacting substance, and histamine release from anaphylactic guinea-pig hearts, and its pharmacological modification.

Prostaglandins (Pgs), slow-reacting substance of anaphylaxis (SRS-A), and histamine were released from anaphylactic isolated perfused guinea pig hearts. Pgs were to the greatest part of the F2alpha-type. PgE2 was found in traces only. Neither PgA2, nor the metabolites 13,14-dihydro-15-keto-PgF2alpha and 13,14-dihydro-15-keto-PgE2 were detected in the perfusates. Isoproterenol reduced the PgF2alpha output significantly. This effect was increased by the addition of theophylline. Propranolol did not reverse the effect of isoproterenol, but in a high concentration (5 mug/ml) reduced the PgF2alpha output for its own. Indomethacin completely abolished the anaphylactic prostaglandin release. The histamine liberation was significantly decreased only by the combination of isoproterenol and theophylline, and also by a high concentration of propranolol (5 mug/ml). In contrast to the Pg release, the anaphylactic SRS-A and histamine liberation was not abolished by indomethacin, but rather increased. The results are discussed in view of the possible role of the released substances in the functional events of cardiac anaphylaxis.

Protein kinase and its regulatory effect on reverse transcriptase activity of Rous sarcoma virus.

We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) on reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity of Rous sarcoma virus. Protein kinase from Rous sarcoma virus-transformed chick embryo fibroblasts was purified by DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectric focusing. Purified reverse transcriptase from Rouse sarcoma virus was preincubated with protein kinase and ATP under conditions allowing incorporation of phosphate into substrate protein. After the preincubation, reverse transcriptase activity was assayed in the presence of poly(rA).oligo(dT) as template. A 2- to 5-fold increase of reverse transcriptase activity was found after the preincubation of reverse transcriptase with protein kinase and ATP. Incubation of reverse transcriptase with heat-treated, inactive protein kinase and ATP had no effect on transcriptase activity. When the transcriptase preparation was incubated with protein kinase and [gamma-32P]ATP and subsequently purified by chromatography on phosphocellulose and Sephadex gel filtration, significant amounts of 32P-labeled proteins were found in the fractions exhibiting reverse transcriptase activity, suggesting 32P incorporation into transcriptase or transcriptase-associated proteins. A 20-60% decrease of reverse transcriptase activity was observed after incubation of reverse transcriptase with phosphatase. The results suggest that phosphorylative modification of reverse transcriptase may be critical in the regulation of reverse transcriptase-catalyzed DNA synthesis.

Incompatibility at irrelevant H-2 specificities augments in vivo stimulation of alloaggressive cells.

Lymphoid cells of mice were sensitized in vivo either by H-2 strain-specific tumor allografts or by activation in lethally irradiated F1 hybrids and tested for cytotoxicity on 51Cr-labeled target cells. The release of 51Cr varied linearly with the logarithm to the proportion of effector lymphocytes to target cells and with the time of interaction. The release of 51Cr was immunologically specific and restricted to H-2 incompatibility. Spleen cells immune to public specificities of the target genotype were not cytotoxic. However, lymphoid cells immune to only one private specificity of a third-party target genotype were highly cytotoxic. The cytotoxicity of activated thymus cells on target cells sharing one private specificity with the genotype used for sensitization was significantly enhanced when the effector thymocytes were activated also against H-2 specificities not shared by the target strain. The results suggest that gene products that facilitate sensitization of effector cells may be determined both by the H-2K and the H-2D end of the H-2 complex. It remains to be shown whether the products of these loci, operating during sensitization in vivo, are body-wide correlated to the lymphocyte-defined specificities detectable during the mixed leukocyte culture interaction.

A four-year evaluation of the chronic toxicity of megestrol acetate in dogs.

A 4-year evaluation of the chronic toxicity of megestrol acetate in dogs is reported. .01, .1 or .25 mg of megestrol acetate/kg/day or .25 mg of chlormadinone acetate/kg/day was administered orally for 4 years t o female beagle dogs. The hormone-treated dogs tended to gain more weig ht than did the controls (controls vs. .25 mg megestrol acetate every month after the 3rd p less than .01). All treated dogs revealed decreased evidence of estrus. Mucoid vaginal discharges were more prevalent among the middle and high dose groups. Mean hemoglobin, packed cell volume and total erythrocyte values were slightly decreased while mean total leucocyte count and erythrocyte sedimentation rates were slightly increased in the middle and high dose groups. Clotting me chanism did not reveal any disturbances. Evidence of diabetes consistin g of bilateral cataracts, elevated serum glucose concentrations and glycosuria after 4 years in 2 of 16 high-dose megestrol acetate and in 6 of 15 chlormadinone acetate-treated dogs was revealed. It is concluded that the effects of megestrol acetate were similar but less severe than those of chlormadinone acetate.

Massive prostatic hypertrophy.

An unusual case is presented in which an enormously enlarged prostate gland, producing minimal urinary tract symptoms and changes, was removed via the retropubic route without operative complications and with a benign postoperative course. Applicability of the retropubic procedure in such cases is emphasized. A brief review of the literature is tabulated.

B lymphocytes, T lymphocytes and phytohaemagglutinin responsiveness in atopic dermatitis.

The spontaneous binding of sheep erythrocytes to human lymphocytes with the formation of rosettes was used as a measure of thymus-derived lymphocytes and the rosette formation with complement-coated sheep erythrocytes as a measure of bursa-dependent lymphocytes in the peripheral blood of patients with atopic dermatitis. Both percentage proportion and the absolute number of thymus-derived lymphocytes were slightly but significantly reduced. In a few patients with atopic dermatitis the percentage and absolute number of bursa-dependent lymphocytes were increased. However, when all values were considered, no significant differences were demonstrated. The responsiveness of cultured lymphocytes to phytohaemagglutinin was normal when the culture medium contained foetal calf serum. The response of normal lymphocytes was not impaired by serum from patients with atopic dermatitis. However, the response was depressed when atopic dermatitis lymphocytes were cultured in medium containing autologous serum, indication that a partial lymphocyte defect becomes manifest only in the presence of a serum factor, presumably IgE.

Serum alpha-foetoprotein as a marker for endodermal sinus tumour (yolk sac tumour) or a vitelline component of "teratocarcinoma".

The correlation between serum alpha-foetoprotein (AFP) and the clinical pathological finding of 24 germ cell tumours arising from the testes (14 cases), the ovaries (3 cases), the mediastinum (3 cases), the retroperitoneal region (2 cases), and the sacrococcygeal region (2 cases) are presented. Irrespective of marked differences in age and sex of the patients, primary site of the tumours and clinical outcome, the 24 cases constituted a homogeneous group in fundamental histological patterns and in AFP synthesis. In all cases of endodermal sinus tumour or teratocarcinomas with a distinct vitelline component an increased serum AFP concentrations was found in the pre-operative serum samples. AFP was also demonstrated in the tumour tissue by quantitative determination of AFP in tumour homogenate (5 cases) and, by immunofluorescence technique, positive staining of the cells lining the endodermal sinuses and of the hyaline globules was found (3 cases). In 12 germ cell tumours without vitelline components in the tumour tissue sections, a normal AFP concentration below 20 mug/1 was found in preoperative serum samples.

Hepatitis B antigen - an incomplete history.

Hepatitis B virus still cannot be grown in an in vitro system; therefore, research into hepatitis B antigen (HB Ag) is limited to laboratory methods such as serology, electron microscopy, and biochemistry. These have established the presence of two distinct antigenic components of HB Ag, that associated with the small forms and the outer covering of the Dane particle (HBs Ag), and that of the Dane particle core (HBc Ag). Current findings make it almost certain that the Dane particle represents the hepatitis B virus (HBV), the smaller forms of the antigen representing excess viral lipoprotein. The group of individuals positive for the antigen are considered and the immunopathology of the disease is discussed. Present understanding of the antigen has made it possible to consider the use of HBs Ag as a means of vaccination. However, further information on the immune mechanisms associated with HB Ag are required before this can be accepted as a general means of protection. Finally, looking at currently available techniques, it would appear that passive hemagglutination and radioimmunoassay are both highly specific and sensitive methods of screening for HB Ag.

Nonspecific reactions with the intradermal test for hydatidosis in persons with other helminth infections.

A partially purified Echinococcus antigen solution, prepared by boiling hydatid cyst fluid, was used in the intradermal test for hydatid disease in a Peruvian population. An unexpectedly high rate of positive reactions and poor agreement with serological tests suggested the presence of some agent(s) which produced cross-reactions with Echinococcus granulosus antigens in the intradermal test. Testing of hospital patients infected with a variety of helminths demonstrated nonspecific reactions in persons with Hymenolepis nana, Taenia spp., and Fasciola hepatica infections, mixed parasitisms, and several non-helminth pathological conditions. The findings contraindicated the use of the intradermal test for epidemiological surveys of hydatid disease. It is pointed out that intradermal test positivity rates cannot be used as synonymous with infection prevalence and regional differences do not necessarily reflect differences in the prevalence of E. granulosus infection. The greater specificity of some serological tests favor their use for epidemiological purposes.

Experimentally induced canine toxocariasis: laboratory examinations and pathologic changes, with emphasis on the gastrointestinal tract.

Eght dogs were orally superinfected for 1 month with 50,000 embryonated Toxocara canis ova. Results of laboratory examinations during inoculation and for 2 weeks postinoculation revealed moderate leukocytosis, marked absolute eosinophilia, hypoalbuminemia, increased concentrations of serum glutamic pyruvate transaminase, and in 2 dogs, precipitating humoral antibodies. Other changes were moderate ascites, hepatomegaly, lymphadenopathy, and focal lesions (0.5 to 3.0 mm) in liver, lung, kidney, intestine, abdominal lymph node, heart, diaphragm, and spleen. Microscopically, focal eosinophilic gastroenteritis was produced. Eosinophils and globule leukocytes were increased throughout the intestinal mucosa. Eosinophil-infiltrated and granulomatous lesions were in the same organs listed as having focal lesions, as well as in the pancreas. The importance of serum beta-globulin content as a potential diagnostic tool was emphasized, and the experimentally induced infection was compared with naturally occurring eosinophilic gastroenteritis in the dog.

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha.

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorphia has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop growth on or methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H2O2 dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H2O2 dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H2O2 was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H2O2 for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.

Relation between infarct size and ventricular arrhythmia.

In order to determine whether ventricular arrhythmia is quantitatively related to infarct size estimated enzymatically we studied 31 patients with acute myocardial infarction without cargiogenic shock. Infarct size index was estimated from hourly serum creatine kinase (CK) changes during periods of 48 to 72 hours. Ventricular arrhythmia was quantified by automated analysis of continuous electrocardiographic recordings over a period of 20 hours with the use of the Argus/H computer system. Patients were classified into three groups according to infarct size index. Patients in all groups had similar average heart rate, blood pressure, serum potassium, and arterial pH and PCO2 values during the first 10 hours after admission. The total number of ventricular ectopic beats (VEB), frequency of couplets, and ventricular tachycardia, and peak rate of ventricular ectopic beats during the first 10 hours after admission were all related to infarct size index. For example, patients with small, medium, and large estimated infarct size averaged 26, 104, and 405 ventricular ectopic beats, respectively. These results suggest that the severity of ventricular arrhythmia early after myocardial infarction is related to the extent of myocardial injury as estimated enzymatically. Thus the apparent efficacy and therefore the evaluation of antiarrhythmic agents early after myocardial infarction may be influenced by the magnitude of injury sustained by the heart.

Mode of inhibition of herpes simplex virus DNA polymerase by phosphonoacetate.

Phosphonoacetate is a highly specific inhibitor of herpes simplex virus-induced DNA polymerase. Sensitivity of herpesvirus type 1 or type 2 induced DNA polymerase to the drug was similar. However, DNA polymerases from other sources such as the host cells (Wi-38), Micrococcus luteus, and hepatitis B virus were highly resistant. In addition, Escherichia coli RNA polymerase and reverse transcriptase of Rous sarcoma virus were also insensitive to the drug. Enzyme kinetic studies showed that inhibition was noncompetitive with respect to deoxyribonucleotide triphosphates. The Ki value was about 0.45 muM. The apparent Km values for dTTP, dATP, dCTP, and dGTP were 0.71, 0.75, 0.42, and 0.39 muM, respectively. The base composition of template has no profound effect on the extent of inhibition. The drug caused uncompetititve inhibition with respect to template which indicated that phosphonoacetate did not bind directly to template DNA. Results are presented which suggest that phosphonoacetate did not affect the formation of the enzyme-DNA complex but probably inhibited the elongation step of DNA polymerase reaction.

A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons.

The phenomenon of retrograde intraaxonal transport of extracellular markers introduced at the level of the axon terminal has been suggested as a possible mechanism of communication between the axon terminal and the neuron cell body. We tested the hypothesis that communication after axotomy might consist of a change in the rate of uptake or of transport of material by injured neurons. Small lesions were made with a needle in one retinal quadrant of chicks and immediately afterwards horseradish peroxidase (HRP) was injected into the vitreous body of the eye. The amount of HRP accumulated by some of the neurons of the isthmo-optic nucleus (ION) which project to the damaged area was clearly different from that of nearby cells which project to the non-damaged portions of the retina. The uninjured cells accumulated enzyme marker beginning at 3.5 h after injection. The injured neurons did not accumulate significant amounts of HRP until between 4 and 6 h after injection. Between 6.75 h and 18 h the injured cells in the ION accumulated greater amounts of HRP than cells in other regions, but by 24 h the cells of the ION in the region of injury contained distinctly less label. This pattern of enzyme accumulation was confirmed by counts of the number of HRP-positive granules within cells of chicks fixed 4, 11.75, 12.25, 27.6 and 72 h after injury. In another series of experiments, the axon terminals of the ION were first exposed to HRP, and 1 h later some of the axons were damaged with a needle. In these cases, there was no difference between the injured and control neurons in the time of first appearance of labeled cells in the ION within the first 4 h after injection of HRP. These findings suggest that injury initially results in a decrease in the uptake of the marker rather than a decrease in the rate of retrograde transport. The amount of marker found in the injured neurons later is greater than that found in the control neurons. This subsequent difference may represent an increase in the rate of uptake, transport, or both or a decrease in the rate of degradation of HRP within the cell body as a response to injury of the axon.

[Immunopathology of a model inflammation].

A series of 32 healthy individuals has been examined, in whom a large number of model inflammatory lesions of the "skin window" type had been produced. Physiological salina solution and diptheriac toxoid were administered by dropping as well as by intradermal injection into the lesioned areas of these individuals. The cover slips were removed after 12, 24 and 48 hours. The control inflammation without antigenic stimulation was characterized by the presence of neutrophilic granulocytes, monocytogenic macrophages and, later, also by epitheloid cells and foreign body giant cells. Celluization of the immune conditioned inflammation mainly consisted of rather small mononuclear cells related to the lymphatic series on the basis of their morphological and cytochemical features. In addition, neutrophils were also found, as well as typical macrophages, epitheloid and foreign body gaint cells. In the 24 and 48 hour preparations, particularly in those obtained from lesions following intradermal antigen administration, basophilic and eosinophilic granulocytes were a regular finding. The ftndings show a variability of the mutual proportions of immunocompetent and immunoneutral cells in the inflammatory cellulization under various condition.

beta2-Microglobulins.

A low-molecular-weight protein, beta2-mu, isolated from urine and other biological fluids was shown to have an amino acid sequence related to constant regions of the immunoglobulin heavy chain--to CH3 comain in particular. Several homologues of this protein were also isolated and partially sequenced. The protein was shown to be localized in the membranes of nucleated cells and found to be associated with HL-A antigens. Its biological and/or immunological function is still unknown; however, it binds to complement and is cytophilic to guinea pig macrophages as well as to other cells. Antibodies to beta1-mu can act as mitogens and can blcok stimulation in MLCs but not recognition by "killer" lymphocytes.

A temporal component of the auditory evoked response.

We studied the 75-225 msec portion of the auditory evoked response (AER) in 32 normal adults at vertex (Cz) and temporal (T3 and T4) placements referred to a balanced, noncephalic reference electrode using a monaural 1 msec click stimulus delivered every 4.7 sec at 60 dB above threshold. The tape-recorded EEG was filtered at 1-25 c/sec, and 128 individual responses were summed, sampling every 0.5 msec for 250 msec post-stimulation. The Cz AERs showed the classic vertex response, a negative peak, N1, at 100 msec, followed by a positive peak, P2, at 160-200 msec. The T3 and T4 AERs were similar to the Cz AERs from 0 to 80 msec and from 200 to 250 msec. They differed significantly from the Cz AERs from 80 to 200 msec. The difference is best explained by the hypothesis that the Cz AERs consisted of N1P2, while the T3 and T4 AERs consisted of N1P2 plus an additional superimposed component, which we called the T complex, comprising a positive peak, Ta, at 105-110 msec, and a negative peak, Tb, at 150-160 msec. By computer, the corresponding Cz and T3 or T4 AERs were normalized to equalize their amplitudes, and the former was subtracted from the latter, thus isolating the T complex. The Ta peak was found to occur 1.5 +/- 1.6 msec earlier at the electrode contralateral to stimulation, and 2.2 +/- 4.0 msec earlier at the T4 (right) electrode. Both differences were statistically significant. The T complex amplitude was greater at the electrode contralateral to stimulation and at the T4 electrode. These findings appear to resolve current controversies concerning the form of the temporal AER. While N1P2 is apparently a product of widespread areas of cortex, we conclude that the T complex is probably a product of secondary auditory cortex.

The sleep of the baboon, Papio papio, under natural conditions and in the laboratory.

The sleep pattern of sixteen baboons (Papio papio) was studied under two very different conditions: (1) in a laboratory at Marseilles, the monkey being immobilized in a restraining chair in a soundproof cubicle; (2) in an African reserve, the monkey being housed in a large cage placed in its natural environment. Some very marked differences emerged. Sleep in the laboratory was longer (by 24 min) and richer in stage 3 and paradoxical sleep. In Africa, however, the sleep showed much more stage 1, was more fragmented and stages 2 and 3 and paradoxical sleep episodes were of shorter duration. Records made in Africa indicate that sleep is independent of slight environmental changes (day length, brightness of the moon, variations in temperature, calls of predators). But the comparison of the two series of results reveals the reorganization which occurs when the monkey is exposed to such different conditions. This adaptation to the environment affects, unequally, the various slow sleep stages and paradoxical sleep. In fact, the major modifications occur in stages 1 and 3 of slow sleep and in paradoxical sleep, while stage 2 appears to constitute the stable, unmodifiable nucleus of sleep.

[Treatment of burns].

Bibliographic data on pathophysiology, therapeutic methods and mortality rate of burn disease are compared with own clinical experiences with a standardized therapy. Within a period of 18 month 35 patients with burn disease of special therapeutic problems were treated besides the overall number of patients in the operative intensive care unit of the Klinikum Steglitz. As special devices for patients with burns still do not exist provisional solutions had to be developed. It is referred to the close connexion between prognosis and mortality, on the one hand, and the age of the patients and the degree of burns, on the other hand. Whereas shock treatment, medicamentous therapy and nutrition are largely standardized, there are strongly differing opinions on local treatment and surgical wound care. The routine application of gammaglobulines, antibiotics and heparin controversial, too.

Multiple antigenic sites on an eicosapeptide. I. Precipitin studies in the goat.

Purified peptide 105-124, an antigenic determinant from the carboxy terminus ribonuclease, was found to form an immune precipitate with antibody to that region prepared by affinity chromatography from goat hyperimmune antiserum to reduced carboxymethylated ribonuclease (CM-RNase). Cm-rnase also gave an immune precipitate with the antibody. Purified antibody to another region of similar size (40-61) did not form a precipitate with CM-RNase but did co-precipitate in the presence of antibody to peptide 105-124 and CM-RNase. The precipitin reaction between antibody to peptide 105-124 and CM-RNase was inhibited by two synthetic derivatives, peptides 118-124 and ala114-RNase 114-124. Stoichiometry of the precipitin reactions of antibody to 105-124 with CM-RNase or peptide 105-124 suggested an antigen valency of three or more. Consistent with this both peptides 105-124 and ala114-RNase 114-124 elicited immediate cutaneous reactions but 118-124 did not. These findings suggest that the eicosapeptide 105-124 is multivalent since at least three antibodies can react simultaneously with it.

Selective and non-selective lymphocytotoxicity in human melanoma: observation on the effect of long-term culture and fetal bovine serum on target-cell sensitivity to lymphocytes.

In vitro cell mediated cytotoxicity (CMC) assays have been conducted in a human melanoma system with a 3H-proline retention technique. Melanoma target cells from long-term cultures ("cell lines") are found to exhibit increased susceptibility for lymphocyte cytotoxicity in comparison to the same target cells from short-term culture. The higher sensitivity of the "cell line" derived target cells is seen with lymphocytes, irrespective of diagnosis of the donor. In parallel experiments with the target cells grown in medium supplemented with fetal calf serum (FCS) and AB+ human serum (from a normal male doner), the melanoma target cells grown with FCS do not show any enhanced cytotoxicity, suggesting no causal relationship of such enhanced sensitivity of "cell line"-derived target cells to "heterologous melanoma antigens" that might have been acquired by the target cells following the use of FCS in tissue culture. In controlled assays of in vitro CMC, lymphocytes from melanoma patients (14/44) exhibited selective cytotoxicity (destruction of only one target-cell type) against the melanoma target cells, whereas only 3/97 control lymphocytes (other malignancies and normal donors) showed such melanoma-selective cytotoxicity. This difference is statistically significant at p less than 0.001. Non-selective cytotoxicity (destruction of two or more unrelated target cell types) was seen with lymphocytes from 9/44 melanoma patients, 13/51 patients with other malignancies and 8/46 normal donors. No correlation of selective cytotoxicity could be established with donors' age, sex, stage of disease, therapy or history of blood transfusion. Such a correlation may emerge as our series becomes larger. Despite the lack of any correlation between selective cytotoxicity and disease status, our study reaffirms the existence of selective cytotoxicity by melanoma patients' lymphocytes against melanoma target cells.

Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.

Characterizaiton of two populations of human lymphocytes bearing easily detectable surface immunoglobulin.

Two separate lymphocyte populations, each bearing easily detectable surface immunoglobulin, have been detected in human peripheral blood. The first, B cells, has surface determinants that are stable at 37degreeC, but are removed by pronase and regenerate in culture. The cells are nylon adherent and have a receptor for C3, and studies with unit gravity sedimentation indicate they are mostly small lymphocytes. B cells comprise 9.5% of the total lymphocytes, with the normal range from 3-16%. As many or more lymphocytes lack membrane-incorporated Ig determinants but have an Fc receptor that binds IgG1 and IgG3 in normal serum maximally at 4degreeC. This receptor for cytophilic IgG is removed by pronase but not by trypsin. The second population has been named L lymphocytes because of membrane-labile IgG determinants. L cells do not adhere to nylon, do not form rosettes with sheep erythrocytes sensitized with antibody and mouse complement, and are larger than small lymphocytes. These lymphocytes with cold-reactive Fc receptors for serum IgG do not form E-rosettes or respond to phytohemaggutinin. Since L cells do not have surface markers of T and B lymphocytes, it is likely that they comprise a separate population.

Histochemical localization of glutathione in tissues.

A histochemical method has been developed for the localization of glutathione (GSH) in frozen sections from various tissues including liver, lung, kidney, testis and eye. The reliability and specificity of the method has been investigated by comparing the rates of reaction in tissue and gelatin sections and after depletion of GSH in liver by diethyl maleate. In principle, the method is based on the formation of an irreversible complex of mercury orange with the --SH group of GSH. A 5-min staining period was found to be optimal for staining the --SH group of GSH. In brief, frozen sections 8 mu thick are stained with a 50 muM solution of mercury orange dissolved in toluene, counterstained in 0.05 per cent methylene blue and mounted in Histoclad. Pretreatment of the sections with fixatives or drying them in air completely prevented the staining. In hepatic lobules the brick red granules of the GSH mercury orange complex were distributed uniformly, whereas in other tissues they were not uniform. The GSH staining was localized in the proximal convoluted tubules in the cortex of the kidney, the interalveolar epithelial cells of lungs, the epididymis and the capsule of testis, epithelial cells of vas deferens and the periphery of the lens.

Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. II. Determination of frequency and characteristics of idiotypic T and B lymphocytes in normal rats using direct visualization.

Anti-idiotypic antibodies made against the antigen-binding receptors of T lymphocytes for a given antigen (Ag-B locus antigens in rats) can be shown to react with IgG antibodies of the same antigen-binding reactivity. Using such anti-idiotypic antibodies, normal Lewis T lymphocytes of B and T type can be visualized by the use of anti-(Lewis-anti-DA) antibodies. Visualization was made possible by the use of direct fluorescent antibody tests or by autoradiography. Using the first technique and naked eye observations 6.2% of normal Lewis T lymphocytes expressed idiotypic markers signifying anti-DA reactivity, whereas anti-DA-reactive B lymphocytes as measured by this approach was in the order of 1.1%. Autoradiography was purified normal Lewis T lymphocytes gave similar figures. When comparing the intensity of fluorescence at the single cell level using quantitative cytofluorometry anti-idiotypic antibodies reactive with T lymphocytes gave a similar degree of intensity as was obtained using anti-Ig antibodies against B lymphocytes.

The major histocompatibility complex determines susceptibility to cytotoxic T cells directed against minor histocompatibility antigens.

Cytotoxic cells were generated by immunizing one strain of mouse with cells from an allogeneic strain which carries the same H-2 region. The effector cells assayed in a 4 h 51Cr release assay were shown to be T cells and indistinguishable, except in specificity, from cytotoxic T cells directed at H-2 alloantigens. Although the genetic differences between responder and stimulator cells responsible for the immunization did not code in H-2, the H-2 complex did restrict susceptibility of target cells. For example, BALB.B cytotoxic cells (H-2b) immunized against and capable of lysing C57BL/6 cells (H-2b) would not lyse B6.C/H-2d target cells. C57BL/6 and B6.C/H-2d are congenic and differ in the H-2 region. Two hypotheses are considered to explain the H-2 restriction of susceptibility to cytotoxic T cells generated by an H-2 identical alloimmunization. (a) The dual (self) recognition hypothesis states that the cytotoxic cell has two recognition units, one for H-2-coded structures and another clonally restricted receptor for the minor alloantigen. (b) The interaction antigen hypothesis states that all the surface alloantigenic determinants recognized by cytotoxic T cells are the result of interaction between H-2- and non-H-2-coded gene products. Two lines of evidence, one with F1 effector cells and the other a cold target competition experiment, are presented which argue strongly in favor of the interaction antigen hypothesis. The regions of H-2 required to be histocompatible were mapped to the D region and to the left of IC, probably the K region. These results, and recent work on the response to virus-infected and TNP-modified syngeneic cells, suggest that cytotoxic cells are restricted in specificity to preferentially recognizing alterations in structures that are coded in the major histocompatibility complex.

Repeatability of the duration of oestrus and breed differences in the relationship between druation of oestrus and ovulation rate of sheep.

The duration of oestrus and the time interval from removal of progestagen-impregnated pessaries to the onset and end of oestrus were examined in Texel, Finnish Landrace, Galway and Fingalway (Finnish Landrace X Galway) ewes. The differences among the breeds in the relationship between these variables and ovulation rate at the controlled oestrus were also investigated. Breed differences were significant for all traits except the interval from pessary withdrawal to the onset of oestrus. The relationship between ovulation rate and both the interval from pessary withdrawal to the onset of oestrus and the duration of oestrus differed significantly among the breeds. The repeatability of the duration of oestrus was significant for Texel and Rambouillet ewes (mean = 0.5) and for pooled data from ewe lambs of various breeds. It was concluded that, in view of the breed differences in the relationship between ovulation rate and duration of oestrus and other traits, generalizations should not be made from among-breed to within-breed relationships. The high repeatability for the duration of oestrus may mean substantial heritabilities for the physiological determinants of oestrus duration.

Immunological comparison of azurins of known amino acid sequence. Dependence of cross-reactivity upon sequence resemblance.

To examine further the dependence of immunological cross-reactivity on sequence resemblance among proteins, we carried out micro-complement fixation studies with rabbit antisera to bacterial azurins of known amino acid sequence. There is a strong correlation (r = 0.9) between number of amino acid substitutions and degree of antigenic difference (immunological distance) among these azurins. The antigenic effects of amino acid substitutions are thus approximately equal and approximately additive. Similar observations and inferences were made before with a series of bird lysozymes. Indeed, the same approximate relationship between immunological distance (y) and percent difference in amino acid sequence (x) holds for both azurins and lysozymes, namely y congruent to 5x. An explanation is given for the dependence of immunological cross-reactivity on sequence resemblance among proteins. This entails reviewing evidence regarding the nature and number of antigenic sites on globular protein antigens as well as evidence for the existence of evolutionary biases against substitutions that are internal or cause large conformational changes. The explanation we give may apply only to those naturally occurring, globular, monomeric, isofunctional proteins whose sequences differ substantially from that of any rabbit protein.

Triiodothyronine and thyroid-stimulating hormone in protein-calorie malnutrition in infants.

Protein-calorie malnutrition (P.C.M.) in a group of 43 Senegalese children aged eighteen to thirty months was characterised by a sharp fall in serum-triiodothyronine (T3) concentration to 25-3% of the mean value in healthy age-matched controls. This decrease in T3 was significantly (P less than 0-001) more pronounced in kwashiorkor of recent onset than in long-term P.C.M., a finding which suggests that impaired thyroxine (T4) monodeiodination in the liver was responsible for the fall in serum-T3 concentration rather than a reduction in the secretion of T3 by the thyroid. Serum-T3 concentrations became normal in both malnourished groups after two weeks of appropriate nutrition. Serum-T3 concentrations in healthy, euthyroid, Senegalese children were higher than in White children. In frank kwashiorkor in Senegalese children, serum-thyroid-stimulating-hormone (T.S.H.) concentrations were within the normal range throughout the entire course of dietary therapy, indicating that the children remained euthyroid. In contrast, protracted P.C.M. led to impairment of the T.S.H./T3 feedback mechanism and to a condition resembling hypophysectomy, which required two weeks' dietary therapy for its correction.

Coeliac disease: a cause of various associated diseases?

Deposition in other organs of immune complexes originating from the small-intestinal musosa is suggested as a possible reason, in some patients, for the described association between coeliac disease and a range of "autoimmune" diseases.

Infantile gastroenteritis: a clinical study of reovirus-like agent infection.

In a clinical study of 32 infants with symptoms from infections with the human reovirus-like agent (R.I.A.) identified by electron microscopy (E.M.) of faecal extracts, a fairly consistent clinical pattern was found in 30 who had a gastroenteritis-like illness. The disease was usually mild, affecting mainly infants less than 2 years and males more commonly than females. The incubation period appeared to be 48-72 hours; and the onset was sudden, often with vomiting in the first 1-2 days of the illness. Loose yellow-green offensive stools without blood or mucus developed after a variable time, and there was often accompanying fever. Severe dehydration and electrolyte inbalance were uncommon; and with standard treatment the illness was uncomplicated, usually lasting 5-8 days. These features resemble those of previously reported winter epidemics of infantile non-bacterial gastroenteritis, and it is suggested that these epidemics were due to R.L.A. 2 infants in whom R.L.A. was identified in the stool did not have a gastroenteritis-like illness although both had protracted diarrhoea.

Treatment of osteoporosis of ageing with 1alpha-hydroxycholecalciferol.

Seven patients with osteoporosis of ageing were treated with synthetic 1alpha-hydroxycholecalciferol (1alpha-H.C.C.) for 3-4 months. The compound was given at a daily oral dose of 2 mug together with an oral supplement of 1 g of calcium. Clinically there was a striking improvement in the patients' physical fitness. Increased bone formation and mineralisation were seen on iliac-crest bone biopsy, and this was supported by an increased osteoblastic activity demonstrated by histochemical measurement of alkaline-phosphatase activity. Bone histology furthermore showed a reduced bone resorption, which was supported by a reduced urinary excretion of total hydroxyproline. Photon absorptiometry of the forearm accorded with the histological findings, showing a significant increase in the bone mineral content. Serum-calcium rose in all patients, one developing a severe transitory hypercalcaemia. The urinary excretion of calcium and magnesium increased significantly. The serum concentrations of 25-hydroxycholecalciferol and parathyroid hormone were not significantly affected by the treatment. It is concluded that 1alpha-H.C.C. is an effective tool in the treatment of senile osteoporosis.

Incongruous referrals.

A survey of 1344 patients registered at a new health centre in Glasgow assessed the prevalence of symptoms and referrals together with subjective, gradings of medical symptoms in terms of pain, disability, and perceived seriousness, and of social symptoms in terms of worry or inconvenience. These grading scales were used to define referral behaviour which appeared to be incongruous in the light of the respondents' own perceptions of their symptoms. In this way incongruous referrals indicated the size of the medical and social symptom "iceberg" and "trivia". For both medical and social symptoms the "icebergs" were larger than the "trivia"; the medical-symptom "iceberg" was two to three times greater than the medical-symptom "trivia".

Localization of laryngeal motor neurons in the kitten.

In a series of 12 newborn kittens, horseradish peroxidase (HRP) was used to trace retrograde axoplasmic flow in the motor neurons to laryngeal muscles. The animals were sacrificed 24 hours after injection of HRP into a specific laryngeal muscle, and the brain stems were stained for peroxidase. This clear-cut colorimetric method permitted the localization of the motor neurons in two nuclei of the ipsilateral brain stem. These are the nucleus ambiguus and the retrofacial nucleus. The primary source of laryngeal motor supply is the nucleus ambiguus. All the laryngeal muscles were represented here in two divisions. Adductor neurons were located in the dorsal division and were more loosely arranged in the lateral reticular formation. The abductor neurons of the posterior cricoarytenoid muscle formed the compact ventral division of the nucleus ambiguus and were fewer in number than adductor neurons by a factor of four to one. Since the expiratory and inspiratory centers are also located dorsally and ventrally in the brain stem reticular formation, the motor cells of the nucleus ambiguus are conveniently arranged to receive their afferent input. This arrangement is probably the result of phylogenetic development of abductor laryngeal function and pulmonary function in lower forms. A second source of laryngeal innervation is the retrofacial nucleus. This small nucleus is situated rostral to the nucleus ambiguus and is made up of small and medium-sized neurons of at least two types. Only the cricothyroid (CT) and posterior cricoarytenoid (PCA) muscles were shown to have significant innervation from this nucleus. The CT neurons were located peripherally while the PCA cells occupied the central portion of the nucleus. The functional significance of this nucleus is unknown, but it is suggested that it may have something to do with the various types of muscle units that have been demonstrated physiologically in the CT and PCA muscles.

A study of the plasma kinin-generating system in children with the minimal lesion, idiopathic nephrotic syndrome.

Although the precise etiologic incitant of the minimal lesion idiopathic nephrotic syndrome of childhood is not known, it is likely that a host mechanism mediates the permeability alterations of the glomerular capillary wall resulting in massive proteinuria. As a first step in examining the possibility that local kinin release may account for the proteinuria in this disorder, two parameters of the plasma kinin-generating system, plasma prekallikrein and kallikrein inhibitor, were assayed during 27 nephrotic episodes in 21 corticosteroid-responsive children. Plasma kallikrein was assayed by means of its esterase activity on a synthetic arginine ester substrate, N-alpha-tosyl-L-arginine methyl ester (TAMe), after activation of Hageman factor by kaolin. This activity, after subtraction of spontaneous arginine esterase activity (i.e., TAMe esterase activity measured in plasma not exposed to kaolin) is derived from prekallikrein. Plasma prekallikrein activity in 11 normal children was 99.6 +/- 2.9 mumol TAMe hydrolyzed/ml plasma/hr (mean +/- SEM). Kallikrein inhibitor was quantified in arbitrary units. Kallifrein inhibitor activity in 11 normal children was 0.94 +/- 0.04 units. During the overt nephrotic syndrome, before initiation of intensive daily corticosteroid treatment, mean values were: prekallikrein, 58.5 +/- 7.24 mumol/ml/hr; and kallikrein inhibitor, 0.35 +/- 0.06 units. After corticosteroid-induced remission occurred, mean values were: plasma prekallikrein, 118.6 +/- 3.2 mumol/ml/hr; and kallikrein inhitor, 0.78 +/- 0.03 mumol/ml/hr. Both parameters were again assayed in 14 of the 21 children after complete cessation of corticosteroid treatment. Plasma prekallikrein was normal, 99.6 +/- 4.8 mumol/ml/hr; but kallikrein inhibitor was still somewhat depressed, 0.84 +/- 0.03 units. A subset of 9 patients had marked depression of plasma prekallikrein to levels less than 20 mumol/ml/hr and essentially undetectable inhibitor activity. Serum alpha-2 macroglobulin was elevated in nephrotic patients: mean value during relapse, 862 +/- 29 mg/100 ml; during corticosteroid-maintaining remission, 615 +/- 29 mg/100 ml. After cessation of corticosteroids, mean serum level was 481 +/- 20 mg/100 ml. The proportional reduction of plasma prekallikrein and kallikrein inhibitor suggested that an enzyme-inhibitor complex formed in vivo, perhaps at a local site of activation in proximity to the glomerular basement membrane. These data suggest that the plasma kinin-generating system may be the host effector mechanism subserving the increased glomerular capillary permeability in the minimal lesion nephrotic syndrome of childhood.

Stimulation and inhibition of anti-hapten responses in guinea pigs immunized with hybrid liposomes.

Guinea pigs were immunized with liposomal model membranes containing phosphatidylethanolamine (PE) or glycerophosphorylethanolamine (GPE) derivatives in which the amino function was substituted with either dinitrophenylaminocaproyl (Dnp-Cap) or mono(p-azobenzenearsonic acid)tyrosyl (ABA-Tyr) residues. Previous studies have demonstrated that hapten-specific antibodies are elicited by DNP-Cap-PE or ABA-Tyr-PE sensitized liposomes and that cell-mediated immunity is induced by ABA-Tyr-PE (but not Dnp-Cap-PE) sensitized liposomes. These liposomes differ from conventional immunogens in which haptens are covalently attached to immunogenic carriers. This investigation describes two new aspects of liposomal immunogenicity in animals immunized with hybrid liposomes containing both Dnp-Cap-PE and ABA-Tyr-PE. (1) Stimulation of the anti-Dnp response by incorporation of increasing amounts of ABA-Tyr-PE; (2) inhibition of anti-ABA antibody formation by incorporation of increasing amounts of DNnp-Cap-PE. The two phenomena are dependent on the presence of each determinant in the same lipid bilayer. Thus, entrapment of the water-soluble deacylated derivative of ABA-Tyr-PE (i.e., ABA-Tyr-GPE) in a aqueous compartments of Dnp-Cap-PE sensitized liposomes does not enhance anti-Dnp antibody production. Similarly, entrapment of the non-amphipathic derivative of DNP-Cap-PE (i.e., Dnp-Cap-GPE) within ABA-Tyr-PE sensitized liposomes does not suppress anti-ABA antibody formation. Furthermore, mixtures of Dnp-Cap-PE sensitized liposomes and ABA-Tyr-PE sensitized liposomes neither stimulated nor inhibited the anti-hapten responses. These results indicate that preparation of hybrid liposomes with different N-substituted PE derivatives provides an extremely convenient method for controlling hapten and/or immunologic carrier determinant density.

Chemical nature of atrial specific granules.

The specific granules are argentafugic when ultrathin sections of Araldite-embedded atria are stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The entire core of the atrial specific granules is moderately positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate- (GMA-) embedded atria are stained with phosphotungstic acid at a low pH. A similar reaction is shown by the cell coat, intercalated discs, residual bodies (C-granules), and Z-discs, as well as by a very small portion of the Golgi complex. Analogous results are obtained with semithin sections of GMA- embedded atria stained according to the periodic acid-Schiff (PAS) technique. In ultrathin sections of GMA-embedded atria stained with dialyzed colloidal iron (DI), the cell coat of the cardiocytes is positive, unlike all the other cytoplasmic organelles. When ultrathin sections of GMA-embedded atria are incubated with proteolytic enzymes (pronase, pepsin, or trypsin), atrial specific granules and Z-bands and, to a much lesser degree, cell coat and sarcolemma are selectively digested. Proteins are also distinctly demonstrated in the paranuclear specific granules by a variety of histochemical techniques. These results indicate that atrial specific granules are rich in proteins and possess a weak complement of complex carbohydrates.

Contamination of anti-immunoglobulin reagents with antibodies to beta 2-microglobulin and other unrelated antigens. Effects on immunofluorescence staining of human lymphocytes.

IgG fractions from three of four rabbit antisera to Bence Jones proteins of chi-type were found to contain antibodies to beta 2-microglobulin and to stain 80%-100% of human blood lymphocytes by indirect immunofluorescence. Antibody fractions from these sera, which contained anti-beta 2-microglobulin but not anti-Ig, stained all lymphocytes, whereas the isolated anti-Ig antibodies (anti-chi) stained only a minor cell population. In both instances, the specificity of the staining was confirmed by absorption experiments. One antiserum to the constant half of lambda-type Bence Jones protein also contained antibiodies to beta 2-microglobulin and stained all lymphocytes. Four other anti-lambda reagents contained no antibodies to beta 2-microglobulin and stained at most about half of the lymphocytes. The antigen responsible for this staining is unknown. The isolated anti-immunoglobulin antibodies (anti-lambda) stained only 5%-10% of the lymphocytes. Antisera to serum IgG or its fragments were free of antibodies to beta 2-microglobulin and stained only 10%-25% of the lymphocytes. This staining was in all instances due to antibodies to human immunoglobulin. Five of eight undiluted sera from normal rabbits with no detectable antibodies to human immunoglobulin or beta 2-microglobulin stained 25%-60% of the lymphocytes. This staining rapidly disappeared on dilution.

LD antigens associated with HL-A8 and myasthenia gravis.

The finding that the SD antigen HL-A8 is associated with myasthenia gravis has raised the question as to whether or not there is also an association between some specific LD antigen and myasthenia gravis and/or HL-A8. MLC technique was used to study LD antigens in 33 myasthenic patients. The cells of a myasthenic patient who was homozygous for both HL-A and LD products were used in MLC tests as stimulators with HL-A8 bearing cells from 24 myasthenics, their 17 relatives and 16 controls and to non-HL-A8 cells from nine myasthenics and 16 controls. At least three different LD genes were found to be associated with HL-A8. One of them, called LDm' was present in 17 (63 %) of the 27 HL-A8-bearing haplotypes of myasthenics, and in nine (47 %) of the 19 HL-A8-bearing haplotypes of controls. In our study LDm was not found in the 84 haplotypes devoid of HL-A8. LDm is strongly associated with HL-A8 and through this also with myasthenia. Calculated from phenotype frequencies for HL-A8 and its association to LDm' the LDm frequencies are 9 % in the control population, 30 % in myasthenics and 48 % in young females with onset of myasthenia below 35 years. LDm had no indendent correlation with any of the clinical parameters of myasthenia gravis, even though is was found more often in HL-A8+ females with the onset of myasthenia before 35 years than in the whole myasthenic group. LDm appears to be similar to LD8a antigen.

Short-term and long-term effects of plasmapheresis on serum proteins and immunoglobulins.

In order to evaluate the short-term and long-term effects of plasmaapheresis on serum proteins and immunoglobulins, the levels of alpha1, alpha2, beta, and gamma globulins, and IgG, IgA, and IgM were measured and statistically evaluated in 41 active plasmapheresis donors donating 500 to 1,000 ml of plasma weekly for up to three years. During the initial four months of plasmapheresis, the percentage of alpha1 and alpha2 globulins manifested a statistically significant rise and the IgG, IgA, and IgM concentrations declined. By the end of ten months, only the IgM continued to be depressed. Although the concentration of IgM continued to show a statistically significant decline for three years, it remained well within the normal range of values for our laboratory. Although no statistically significant difference existed between the baseline value of albumin and the level reached at the end of the third year, a gradual rise was followed by a decline in this interval. Most of the statistically significant alterations of serum protein and immunoglobulins occurring in plasmapheresis donors are seen in the initial six months of plasmapheresis. A falling serum protein in this time period is most likely an indication of declining immunoglobulins. It is feasible and appropriate to measure the donor's total serum protein at the time of each plasmapheresis. Any untoward reduction in this value necessitates quantification of the serum immunoglobulins. Routine measurement of the immunoglobulins in the face of normal total serum protein can be performed on a less frequent basis as is presently recommended by accrediting agencies, although this study should be performed more often during the first six months of a serial plasmapheresis program.