Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.


The endogenous reverse transcriptase activity of Gibbon ape lymphoma virus: characterization of the DNA product. The DNA product of the endogenous reverse transcriptase reaction of Gibbon ape lymphoma virus has been analyzed and characterized. Data show that in simultaneous detection assays in which the type and/or concentration of divalent cation is varied the best yield of rapidly-sedimenting DNA was obtained from reactions containing 1.5 mM Mn2+. This yield is ten-fold better than the yield observed at the optimal Mg2+ concentration (5.0mM). Evidence is presented to show that DNA synthesized at the optimal concentration of either of these cations consists of large pieces varying in size from 4 to 12S. This DNA hybridizes efficiently to homologous viral RNA (greater than 60 percent annealing) and protects at least two-thirds of GALV 70S [32P]RNA from ribonuclease digestion. The hybrids formed with homologous viral RNA are stable as evidenced by their thermal elution patterns from hydroxylapatite columns. In contrast, DNA synthesized in reactions in which the concentration of Mn2+ or Mg2+ was greater than optimal was predominantly 4S or smaller in size and displayed a low level of hybridization (less than 10 percent) to homologous viral RNA.


[Evaluation of cytochemical methods of studying the localization of alkaline phosphatase in B. subtilis]. A study was made of a possibility of using the "lead" and the "calcium" methods for the determination of localization of alkaline phosphatase in the bacterial cells. Cytochemical medium containing lead nitrate could not be used for determination of the true localization of the alkaline phosphatase in the microorganisms.


[Mechanism of the natriuretic and diuretic action of neurohypophyseal hormones]. Administration to rats against the background of spontaneous urine excretion of pituitrine, vaso pressin and oxytocine caused a diuretic and natriuretic effect on account of the reduction of the tubular reabsorption. In rats with removed adrenal glands the preparations under study did not produce any diuretic influence, and natriuresis was observed only in administration of vas opressin. The diuretic effect of the preparations was absent against the background of hypophysectomy, whereas the natridiuretic effect was retained. It is supposed that the diuretic influence of the neurohypophyseal hormones was associated with the activation of the hypophysis-adrenal system, and some other additional mechanism took part in the natridiuretic effect.


Antinuclear antibodies during procainamide treatment and drug acetylation. Acetylator capacity was determined in two groups of patients who had received procainamide for more than three months. In seven patients antinuclear antibodies (A.N.A.) were detected during treatment, and these changes disappeared (in six patients) or were less pronounced (one patient) after withdrawal of the drug. These patients tended to have faster acetylation rates, and five were phenotypically "rapid" acetylators. Five patients who did not develop A.N.A. during treatment had less rapid (P less than 0.05) rates of acetylation, and four were "slow" acetylators. We suggest that the immunological changes which may occur during procainamide treatment may be associated with the acetylated metabolite of procainamide rather than the parent compound and that it might be possible to identify patients at risk.


Isolation of axonally transported glycoproteins with goldfish visual system myelin. [3H]fucose labeled glycoproteins which are rapidly transported in the goldfish optic nerve have been isolated with purified preparations of goldfish optic tectal myelin. SDS acrylamide gels of myelin proteins isolated one day after intraocular injection of fucose show a broad distribution of radioactivity among high molecular weight proteins. At progressively longer times after injection there is a shift in the distribution of radioactivity with the buildup of a peak of label with electrophoretic mobility corresponding to a molecular weight of approximately 50,000. There is no corresponding peak of radioactivity in gels of total tectal membranes.  Gels of myelin labeled through intracranial injections of fucose also show no single prominent peak of radioactivity. Results are discussed with reference to possible mechanisms for the shift in radioactivity patterns on gels and the possible functional significance of the myelin-associated glycoproteins.


Neuronal fibrillar proteins and axoplasmic transport. Proteins synthesized in ganglion cells of the rabbit retina were labeled with [3H]leucine or [14C]glycine injected into the vitreous. The labeled proteins that were transported into the optic nerve and tract were dissolved and analyzed by polyacrylamide gel electrophoresis. Under simple electrophoretic  conditions the incorporated label appeared to be broadly distributed over the bands of axonal proteins but with a discontinuous buffer system where higher resolution was achieved, the prominent stained bands of actin, tubulin and neurofilament did not coincide with any peaks of radioactivity. Thus the specific activity of those proteins is lower than that of other migrating proteins and it is concluded that these proteins do not move in toto with axoplasmic flow.


[Effect of hypothalamic implants changing monoamine levels on continuous estrus in rats induced by androgen treatment in the neonatal period]. The constant estrus induced by neonatal androgenization in the female rat may be disrupted following a drug-induced change of the monoamine levels in the preoptic area and arcuate nucleus of the median eminence. The most precise effect is obtained with 5-HT. This is in favour of a predominant alteration of the serotonergic system which seems to be involved in the sexual differentiation of the brain.


[Effect of hemolysin from Pseudomonas aeruginosa on cell cultures]. Hemolysin of Pseudomonas aeruginosa has a cytopathic action on blood and tissue culture cells. Lysis and disintegration of the architecture of the cell involving membrane and cytoplasm were demonstrated by morphological changes. The hemolytic activity of hemolysin is inhibited by normal sera and by albumin; inhibition is also observed in the action of hemolysin on K.B. cells, but this inhibition is not complete. The hemolytic and cytopathic actions are explained by assuming that they alter the molecular architecture of the membranes. The variability may relate to the differing availability of reactive sites on the cells, or indicate that the two activities are associated with two different enzymes.


[Incorporation of 3H-thymidine in Wistar rat embryos treated with AY 9944]. Intake of tritiated thymidine by the embryos of Wistar pregnant rats treated by AY 9944 is significantly lowered. This decrease is parallel with the embryonic hypotrophy. Is there a causal relationship between the two phenomena?


Identification of the sex chromosomes in the bald eagle. Karyotypes of five American bald eagles (Haliaeetus leucocephalus and H. alacanus) are compared. All had 2n=66 chromosomes which fell into 3 size groups: A, 20 pairs of biarmed chromosomes; B, 9 pairs of acrocentric chromosomes and C, 4 pairs of microchromosomes. C-banding was done in two eagles and a heterochromatic W chromosome was identified in a presumptive female. The ZZ and ZW chromosomes could be identified in the karyotypes.


Insect chromosome banding: technique for G- and Q-banding pattern in the mosquito Aedes albopictus. A modified technique is described for the production of clear G- and Q-bands of somatic metaphase chromosomes of the mosquito, Aedes albopictus Skuse.


Palliation in the management of laryngeal cancer. Comments on the radiotherapeutic approach to advanced disease. The number of patients in the category of advanced cancer of the larynx and other sites will increase as the population increases, as longevity increases, and as increasingly effective treatment prolongs the life of the uncured cancer patient. Such patients expect no lesser effort on their behalf than is offered to patients with clinically curable cancer and an informed public may not be satisfied with the implications of palliation. Any planned improvement in the patient's status, any useful time gained, is successful treatment. The only true failure is the complication that results from plundering of tolerance which might be preserved and used to good effect by careful treatment planning.


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.


Role of cell wall topography in conjugation of Schizosaccharomyces pombe. The surface of the zygotes of Schizosaccharomyces pombe was studied by observing fluorescence following primulin treatment. Conjugation occurred only at the poles. Competence to fuse was independent of actual pole growth and pole ontogeny (old or new pole). The cells were competent to fuse throughout the first three-quarters of the cell cycle. Morphological criteria indicate that cell pairs are not synchronized at the moment of fusion. The length of the G1 phases of individual conjugating cells apparently ranges from 0.1 to 0.7 of the cell cycle duration.


Trichosporon sepsis and leukemia.

Trichosporon sepsis and leukemia. Trichosporon cutaneum is a fungus known to cause superficial nodules over the distal third of hair shafts, mainly scalp hair, and to produce a clinical entity known as piedra. This superficial mycosis occurs mostly in temperate and tropical regions and is rarely seen in North America. Trichosporon cutaneum spesis is described here in a 12-year-old boy with acute lymphocytic leukemia in relapse. To our knowledge this is the first case reported in the literature. Emphasis is made of the increasing rate of fungal diseases as well as of "opportunistic" infections in this type of immunosuppressed patient.


Enhanced cell killing by bleomycin and 43 degrees hyperthermia and the inhibition of recovery from potentially lethal damage. The effect of hyperthermia on bleomycin (BLEO) toxicity and repair was studied in "unfed" monolayer cultures of Chinese hamster cells. Synergy of toxicities was observed with simultaneous exposure to BLEO and 43 degrees. For example, when cells were exposed for 1 hr to BLEO (40 mug/ml) at 43 degrees, survival was reduced to 4 X 10(-5); separately, hyperthermia and 37 degrees BLEO exposure each resulted in a survival of 20%. Heating at 43 degrees prior to drug exposure at 37 degrees also produced substantial sensitization, indicating that the primary sensitizing effect involved cell damage rather than an increased rate of drug action; 41 degrees produced only modest cell sensitization to BLEO and the effect was not retained in cells heated prior to drug exposure. No increase in [14C]BLEO uptake was observed at 43 degrees over than at 37 degrees, and thus the increased cytotoxicity was not correlated with a gross change in cell permeability to BLEO, although increased drug availability to particular sensitive targets could not be ruled out. Studies of the repair kinetics after different 43 degrees BLEO protocols demonstrated that most of the cells sustaining potentially lethal damage rapidly recovered. However, 43 degrees hyperthermia inhibited this recovery and, with increasing durations of 43 degrees exposure, the fixation of potentially lethal damage was enhanced. Because of the substantial repair of BLEO damage observed in vivo, the possible usefulness of hyperthermia as an adjunct to BLEO therapy is discussed.


Demonstration of two molecular variants of carcinoembryonic antigen by concanavalin A sepharose affinity chromatography. The carcinoembryonic antigen (CEA) active glycoproteins from perchloric acid extract of liver-metastasized primary colon tumor have been separated by concanavalin A Sepharose (Con A Sepharose) chromatography. The CEA activities separated by Con A Sepharose chromatography were designated as loosely bound and tightly bound which, respectively, eluted on the Con A Sepharose column between 0.12 and 0.15 M and 0.3 M alpha-methylmannose in a linear gradient of alpha-methylmannose. Further purification of these activities by Sephadex G-200, Bio-Gels A-1.5m and P-300 yielded two variants of glycoproteins (B1 and C2) with CEA activity. Both purified preparations of CEA had similar immunochemical properties. Their A280/A260 ratios were 1.30 and 1.56, respectively. The purified loosely bound CEA (B1) had immunological, chromatographic, and electrophoretic properties similar to those of 125I-CEA, whereas the tightly bound CEA (C2) had a lower molecular weight (120,000 to 140,000). Further, specificity to these two CEA's was established by their reactions in immunoelectrophoresis with preparations of specific goat anti-CEA anti-serum obtained from other investigators. The results indicate the practical use of Con A Sepharose affinity chromatography for the separation and characterization of glycoprotein tumor antigens.


Sequential analysis of transplantable hepatocellular carcinomas. In a previous study multiple characteristics of chemically induced primary hepatocellular carcinomas were described and examined during the initial transplant generations. The present communication reports on these characteristics in subsequent transplant generations followed over a 2-year period. In almost all instances the growth rate, morphology, chromosome composition, and plasma protein and alpha-fetoprotein synthesis of individual tumors have remained relatively constant. However, one spontaneous subline of a diploid tumor demonstrated a sudden extensive rearrangement of its chromosomes simultaneous with a significant acceleration of growth rate. Despite karyotypic evolution, it retained the functional characteristics of diploid tumors, producing no plasma protein or alpha-fetoprotein.


Enhancing effect by metabolic inhibitors on the killing of tumor cells by antibody and complement. Two chemically induced, antigenically distinct guinea pig hepatoma cell lines, line 1 and line 10, which are resistant to killing by rabbit anti-Forssman or specific antitumor antibody and complement, can be rendered susceptible when the cells are pretreated with metabolic inhibitors and drugs commonly used for the treatment of cancer patients. The effect appears within 7 hr after initial contact with the inhibitors and is dependent on temperature and on inhibitor concentration; the effect is reversible within 7 hr, and the process of reversion is also temperature dependent. Not all preparations of tumor cells were rendered susceptible following treatment with inhibitors. In some cases, susceptibility to killing by complement was observed with anti-Forssman antibody but not antitumor antibody. No clear correlation between known metabolic inhibitory activity of the inhibitors and conversion to the sensitive state could be made. The results suggest that properties of nucleated cells, which are under metabolic control, play an important role in the killing efficiency of antibody and complement.


Effect of metabolic inhibitors on the ability of tumor cells to express antigen and bind complement components C4 and C3. Metabolic inhibitors commonly used in the treatment of cancer increase the ability of antibody and complement to kill guinea pig tumor cells in vitro. No correlation was found between increased killing and changes in cell surface antigen concentration or binding of complement components C4 and C3.


Quantitative immunocytochemistry of pituitary receptors for luteinizing hormone-releasing hormone. In Araldite sections of male rat pituitaries, stained after embedding by the unlabeled antibody enzyme method with antisera to native luteinizing hormone-releasing hormone (LH-RH) or LH-RH azo-conjugated to bovine serum albumin, localization is confined mainly to the interior of the large, and to a lesser extent to that of the small, secretion granules of the gonadotrophic cells. Plasma membranes are not demonstrated. Except for weak staining in the granules of corticotrophs, no other pituitary cell is stained. Pretreatment of sections with LH-RH (to dilutions of 4 pg/mul) increases staining intensity in the gonadotrophic granules. Other cells are unaffected. The lesser the gonadotroph staining intensity without pretreatment, the greater the increase (more than 23-fold reactivity). Augmented staining is measurable (P less than 0.001) to antiserum dilutions of 1:240000. Pretreatment with des-Glu-1-LH-RH, porcine corticotropin or rat prolactin has no effect. LH-RH-Gly-10(des-amide) inhibits. Rat glycoprotein hormones enhance staining with anti-azo-conjugated LH-RH. With antinative LH-RH these hormones enhance weak staining, but inhibit strong staining. Thick vibrotome sections of male rat or rabbit pituitaries stained before embedding reveal specific localization on plasma membrane and gonadotrophic secretion granules provided the sections have been pretreated with LH-RH (250 pg/mul). The data show that LH-RH after reaction with receptor is not sterically hindered from binding specific antibodies. Receptor may be found in secretion granules, both in the free state or combined with LH-RH. Plasma membrane receptor, on the other hand, was free under the conditions of the experiments. Immunization with LH-RH elicits not only heteroimmune antibodies specific for LH-RH, but also a group of still ill defined autoimmune antibodies, some of which may conceivably be reactive with glycoprotein hormone alpha-chains.


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.


Ventricular quadrigeminy as a manifestation of concealed bigeminy. Long rhythm strips were analyzed from five patients with frequent ventricular extrasystoles. The predominant pattern was quadrigeminal; i.e., three sinus beats between extrasystoles. However, about 20% of the interectopic intervals contained numbers of sinus beats (S) greater than three. Analysis of the distribution of such values of S greater than 3 revealed that there were many more odd than even values (P less than 0.001). Also, carotid sinus pressure yielded only odd values of S greater than 3. This predominance of odd values strongly suggested the existence of concealed extrasystoles. Therefore, all odd values of S greater than 3 were analyzed to determine whether they satisfied the criterion for concealed bigeminy (S = 2n - 1) or for concealed quadrigeminy (S = 4n - 1). The distribution was found to satisfy the criterion for concealed bigeminy, suggesting that the quadrigeminal pattern was a manifestation of a 2:1 rather than a 4:1 block in a re-entry loop. Stable quadrigeminy occurs often in concealed bigeminy, because the re-entrant impulse finds the myocardium excitable after a normal R-R interval but refractory after a compensatory pause.


Monitoring for serious arrhythmias and high risk of sudden death. Ventricular premature beats (VPBs) are present in 85% of patients with coronary heart disease (CHD). A classification of VPBs based on frequency, multiformity, repetitive pattern, and degree of prematurity promises to aid in identifying patients at high risk of sudden death. Advanced grade or repetitive forms of VPBs are observed in 41% of patients with CHD who are monitored for 24 hours while ambulatory. Exercise stress is less effective than monitoring, but when advanced grades are exposed by exercise, they are likely to be persistent for two or more hours during monitoring. Such persistent and advanced grades characterize patients with multivessel coronary artery disease. Diurnal variations occur in VPB frequency and pattern with significant reduction during sleep. This as well as other facts suggest a primary role for neural activity in the genesis of ventricular ectopy. The relevance of these observations to sudden death is considered.


Ventricular premature beats and mortality of men with coronary heart disease. Frequency and qualitative characteristics of ventricular premature beats (VPB) are determined from 1 hour of electrocardiogram (ECG) monitoring of men with coronary heart disease in a continuing study among members of the Health Insurance Plan of Greater New York. Mortality among 924 such men during an average observation period of 10 months was higher among men with VPB than among those free of VPB. In comparison with the routine 12-lead ECG, the hour of ECG monitoring increased the proportion of men identified as showing ectopic activity from 15% to 52%. Only half of men with 10 or more VPB in the hour of monitoring were noted as having VPB on the routine ECG; mortality among these men was relatively high whether or not VPB appeared on the short sample ECG.


Prognostic grading and significance of ventricular premature beats after recovery from myocardial infarction. A 6-hour taped electrocardiogram recording was obtained prior to hospital discharge on 193 patients less than 66 years of age with a definite or probable myocardial infarction. All ventricular premature beats (VPBs) were identified on the 6-hour recording, and the various VPB characteristics were related to a complicated course (CC) defined as cardiac death or myocardial reinfarction within 4 months after hospital discharge. Only three univariate VPB characteristics (multiform pattern, bigeminal rhythm, and frequency greater than or equal to 20/hr) were significantly associated with a 4-month posthospital CC. By use of bivariate and multivariate combinations of these three VPB characteristics, a two-grade prognostic stratification system was derived: Grade I VPBs (CC = 8%) included patients with less than 20 VPB/hr but with neither multiform nor bigeminal beats: Grade II VPBs (CC = 31%, P less than 0.001) contained patients with greater than or equal to 20 VPBs/hr or with multiform or bigeminal beats or both. Patients with Grade II VPBs had more severe cardiac disease, but when patients were stratified by the clinical severity of their cardiac disease, Grade II VPBs identified patients with higher CC rates. The clinical significance of these findings together with a critical analysis of the prognostic stratification method are discussed.


Ventricular premature beats in a population sample. Frequency and associations with coronary risk characteristics. In a large epidemiological survey, 10,880 men aged 35 to 57 years were screened for ectopic ventricular activity and estimated risk of future coronary heart disease death. A 2-minute lead I electrocardiogram (ECG) was recorded for each man, together with measurement of serum cholesterol, blood pressure, and the number of cigarettes currently smoked. Ventricular premature beats (VPB) occurred in 540 (4.96%) men; of these 459 (4.21%) were uniform, while complex forms were detected in only 7/1000 men. VPB occurring with a frequency of greater than or equal to 10 in the 2-min recording were also rare, occurring in 9/1000 men. No relation was found between the frequency of simple or complex VPB and estimated coronary risk status or between the prevalence of VPB and the individual risk factors of diastolic blood pressure, cholesterol, or cigarette smoking. There was, however, a strong positive association between VPB and the level of systolic blood pressure, and between VPB and increasing age. The lack of association between VPB and overall coronary risk status indicates that myocardial "ischemic" changes in high-risk men may not have progressed sufficiently to alter ventricular excitability or to increase the frequency of VPB.


Arrhythmia prophylaxis: Long-term suppressive medication. The author describes the notions that led to initiation of controlled trials of long-term medication with antiarrhythmic drugs following myocardial infarction, and reviews reports of controlled trials with diphenylhydantoin, alprenolol, and procaine amide. It is concluded that, so far as possible widespread prophylactic use of these drugs to prevent sudden death is concerned, diphenylhydantoin has no place, while procaine amide cannot be evaluated on a large scale because of the frequency with which it causes serious adverse reactions. There is no bar to an extended trial with alprenolol or comparable beta-blocking drugs. The author believes that nonspecific prophylactic long-term use of antiarrhythmic drugs to prevent sudden coronary death outside the hospital is unlikely to prove rewarding.


Application of the patient-self (P-S) test to assess the specificity of hyposensitization with Allpyral-G. Following hyposensitization with Allpyral-G5-grass mix the level of blocking antibody is assessed against the 5-grass mix, against Timothy (one of the grasses) and against Meadow Grass (not included in the five grasses) respectively and the results compared. The implications are that only very limited protection is given against a grass pollen other than those used for the hyposensitization and this confirms laboratory findings that there is minimal cross-reactivity between grass pollen allergens.


A rapid method for the purification and radioimmunoassay of human alpha-fetoprotein. Human alpha-fetoprotein (AFP) was isolated from cord serum on an immunoadsorbent column obtained by covalently linking rabbit anti AFP to cyanogen bromide activated Sepharose. Bound AFP was eluted with 8 M urea with better than 50% recovery. The purified AFP was iodinated prior to its use in a double antibody radioimmunoassay. The purification and radioimmunoassay employ commercially available materials. A standard inhibition curve was obtained which allowed determination of AFP levels between 50 and 100 ng/ml in human serum. The assay was verified by measureing AFP levels in normal female serum, pregnancy serum, cord serum, hepatoma ascitic fluid and a standardized AFP solution.


Treatment of benign tumours of the rectum. The methods of treatment for benign tumours of the rectum are by local removal or local destruction. The benign status of the tumour must be determined histologically or by gross morphology before treatment is instituted. The approaches are either transanal or by posterior proctotomy. The following procedures have been employed with excellent results for the past thirty years at the Ferguson Clinic: 1. Transanally, by local excision, local destruction or snare removal. 2. By the posterior proctotomy approach for benign tumours that could not be removed transanally because of size or inaccessibility. A new 'Two scope technique' first devised in 1969 has been used with great success since.


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.


The influence of C3b inactivator (KAF) concentration on the ability of serum to support complement activation. Minor elevation of the concentration of the C3b inactivator (KAF) in whole serum (by 15-25%) markedly inhibits the capacity of the serum to support complement activation by inulin, aggregated IgG and by low concentrations of CVF. However, there was no effect when large concentrations of CVF were used nor was the spontaneous ageing of C3 slowed by increased KAF concentrations. These findings show that the activity of the C3b feedback cycle can be reduced by raising the KAF concentration above physiological levels. This finding may provide a mechanism for damping down complement activation locally in vivo. It seems that the spontaneous ageing of C3 in vitro does not involve a KAF-inhibitable step and therefore cannot be involved to explain the 'C3 tickover' in vitro.


Accelerated cytodifferentiation of antibody-secreting cells in guinea-pig lymph nodes stimulated by sheep erythrocytes and lymphokines. Lymphokines, produced in response to structurally unrelated antigens, altered the course of a primary anti-sheep erythrocyte plaque-forming cell response within the regional lymph nodes of normal guinea-pigs. Intralymphatic injection of a small dose of lymphokines (0-5-8 mug) 1 day after antigen priming accelerated the rate of indirect plaque-forming cell cytodifferentiation between the 5th and the 9th days of the response. This effect was not related to changes in the level of antigen trapping by lymph node macrophages, but the lymphocyte mitogenic activity may have been important for the response since there was a significant increase in [3H]thymidine incorporation within the lymphokine-treated nodes on the 3rd day following immunization.


Antigenic determinants common to established human B-cell lines, but not shared by human T-cell lines (molt and sommer). Rabbits were immunized with two kinds of human B-cell lines (SOMMER-B, and RPMI number 1788 cells) to get anti-B-cell sera. Sera were absorbed with human red blood cells, human liver, human brain and human T cells (MOLT-4 and SOMMER-T cells). Cytotoxicity of absorbed sera was not only tested against B cells which were used for immunization but also tested against unrelated B cells, including B35M cells derived from Burkitt lymphoma specimens. Results indicated that established human B cells had antigenic determinants which were shared with allogeneic human B cells, but not shared with non-lymphoid cells or tissues, nor with extablished human T cells.


Sister chromatid exchange in human chromosomes from normal individuals and patients with ataxia telangiectasia. A new fluorescence plus Giemsa staining technique now makes the detection of sister-chromatid exchange (SCE) a relatively easy matter in cells containing 5-BrdU-substituted DNA. The technique has been applied to human cells to examine the distribution of SCE between different people and within different chromosomes. The results show: (1) That there were no large differences in the incidence of SCE between blood leukocyte chromosomes from male and female adults and newborn, and that similar frequencies were found in cells from two patients with ataxia telangiectasia which, nevertheless, showed the typical increases in chromosomal aberrations. (2) The distribution of SCE between chromosomes in the complement was found to be proportional to chromosome length, although the smaller chromosomes were under-represented, but not significantly so. (3) The distribution of SCE within chromosomes was nonrandom, with a deficiency in the centromeric and an excess in the mid-arm regions. There was no evidence for an excess of SCE in chromosome regions rich in AT DNA sequences. (4) The frequency of SCE is to some extent dependent of 5-BrdU concentration, but the influence of concentration is minimal within the range of from 1 to 160 muM. Human cells exposed over two cell cycles at these higher BrdU levels have around 14 SCE per cell-a frequency virtually identical with that observed in cultured cells from the Chinese hamster, wallaby, and rat kangaroo.


Sister chromatid exchange and chromosome organization based on a bromodeoxyuridine Giemsa-C-banding technique (TC-banding). Hoechst 33258 fluorescent staining of bromodeoxyuridine substituted chromosomes provided a high resolution technique for following the segregation of replicated chromosomal DNA (Latt, 1973). Modifications have produced the same results after Giemsa staining (Wolff and Perry, 1975). Since this does not necessarily require Hoechst (Korenberg and Freedlander, 1975), we call this bromodeoxyuridine-Giemsa banding (BG-banding). We here describe a further modification which allows one to follow the T-rich strand of the AT-rich satellite DNA of C-band heterochromatin. We call this TC-banding. This technique was used to examine metacentric marker chromosomes found in mouse L-cells that contain many interstitial blocks of centromeric-type heterochromatin in each arm plus the usual two blocks of centromeric heterochromatin. One of the advantages of this technique for such chromosomes is that it is possible to distinguish first from second cell cycle sister chromatid exchange and unambiguously detect centromeric sister chromatid exchange. We found some chromosomes to have high rates of centromeric sister chromatid exchange. After one cycle in bromodeoxyuridine we could examine the satellite polarity of the heterochromatic DNA. Since there was no change in satellite polarity in any of the heterochromatic blocks, marker chromosomes could not have been formed by paracentric inversions, inverted insertions or inverted translocations. These results allow the formulation of several rules of chromosome organization.


[Discontinued treatment with BudR and staining with acridine orange: observation of R- or Q- or intermediary banding (author's transl)]. Discontinued treatments with BudR at different periods of the cellular cycle produce various chromosome banding after staining with acridine orange. In particular, it is possible to observe R- or Q- or an intermediary banding, simply by varying the time of incorporation of BudR. This implies that the amount of AT or GC bases present locally in DNA is not directly responsible for the banding observed. Furthermore it appears that a precise correlation exists between replication and R- or Q-banding: the DNA located at each group of bands replicates either early (R-bands) or late (Q-bands). But these timings overlap towards the middle of phase S: if the treatment is given at that time, it is possible to observe aspects intermediary between Q and R.


[Excessive peripheral conversion of thyroxine (T4) to triiodothyronine (T3) in the pathogenesis of T3-hyperthyroidism (author's transl)]. In a 41-year-old woman and a 3-year-old girl, both of them with T3-thyrotoxicosis, serum levels of total and free T4 and T3 were measured serially during anti-thyroid drug treatment. Attempts to substitute thyroxine during the antithyroid treatment had to be interrupted because the patients became hyperthyroid again with excessive increases in total and free serum T3, even when concentrations of total and free T4 were brought to subnormal levels. The increased conversion of administered thyroxine ceased later on and higher amounts of oral T4 were tolerated after one year of treatment. In both patients there was an extremely low serum T4/T3 ratio, differing in this respect significantly from six other patients with T3-thyrotoxicosis and 41 patients with "conventional" T3/T4-hyperthyroidism. It is concluded that, in patients with T3-thyrotoxicosis due to excessive peripheral T4 to T3 conversion, substitution during antithyroid drug treatment should be either with very low doses of thyroxine or with triiodothyronine in divided daily doses. In such cases the level of serum T3 represents the most reliable biochemical measurement for the control of treatment, serum T4 levels being irrelevant.


Control of snail hosts of bilharziasis in Egypt. 3. Effect of the organophosphorous insecticide, dursban, on carbohydrate metabolism of the snails Biomphalaria alexandria and Bulinus truncatus. The effects of the organophosphorous insecticide, Dursban, on aerobic oxidation, glycolysis, glucose utilization and gluconeogenesis in snails tissues were determined. Dursban had a biphasic effect on the aerobic oxidation of succinate, glutamate + malate and TMPD + ascorbate while it had only an inhibitory action on pyruvate oxidation. The compound significantly inhibited glycolysis, glucose utilization and gluconeogenesis when used at high concentrations (ten times higher than its LC50). However, it had a slight effect on thepreviously mentioned process when its concentration was equal to or approximated its LC50. The relationship between the metabolic effect of Dursban and its molluscicidal activity is discussed.


Kindling effect and sleep organization in cats. A comparative study of sleep organization was made between seven kindled cats and four controls. Longitudinal observation during 5 h of polygraphic recording continued every day at the beginning, irregularly after kindling, for one year. During kindling installation, REM sleep was gradually reduced after each stimulation. When the kindling phenomenon was established each stimulation triggered a typical generalized convulsion and diminution of REM sleep persisted. REM characteristics such as total amount, duration of episode and frequency decreased after each stimulation. Increase of the awake stage was also observed after each stimulation throughout the kindling procedure. These data are compared with other reports which examine sleep organization after generalized convulsions or temporal lobe epilepsy in animal and man.


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.


Far-field recorded frequency-following responses: evidence for the locus of brainstem sources. Two experiments were performed to determine the brainstem origins of the scalp recorded auditory frequency-following response (FFR). The first was a study of FFR onset latency in which responses observed by direct recording from depth electrodes in brainstem auditory nuclei were compared with those obtained from the scalp. The mean onset latency of scalp recorded FFR (5.8 msec) closely approximated the 5.4 msec mean latency of response recorded from within the inferior colliculus (IC). In a second experiment, cooling of the IC greatly reduced or eliminated FFR both within this nucleus and at the scalp. FFR at the medial superior olive was unaffected during cooling. It was concluded that at moderate intensities of stimulation the primary source of scalp recorded FFR is the IC.


The effects of a spirolactone derivative on EEG and cortical single unit activity in the cat. With systemic (60-150 mg/kg) and topical application, Aldactone exhibited strong convulsant action on the cat cerebral cortex. The most common abnormal interictal features of the surface ECoG pattern were sequences of fast waves and slow negative waves associated with positive sharp waves, negative field potentials and burst-like neuronal activation in the extracellular microelectrode record. Another abnormal pattern was a period of ECoG and neuronal inactivity subsequent to the negative transients. These series of sharp and slow waves and of inactivity were interrupted by tonic and clonic ECoG seizures characterized by regular surface positive spikes. Development of seizures, but not of interictal phenomena, could be prevented by intravenous application of Diazepam (1 mg), Nembutal (30 mg/kg) and--less effectively--by Phenhydane (20 mg/kg).


Photic sensitivity of children with absence seizures in slow wave sleep. Consequent upon previous studies on the effect of sleep on spike-wave discharges in absence seizures, photically sensitive spike-wave paroxysms were studied in 4 children with absence seizures, including identical twins, during various sleep stages. Sensitivity to stimuli at all frequencies and intensities was minimal during slow-wave sleep (stages 3 and 4), although photic driving could still be discerned. Trains of paired stimuli were effective in triggering polyspike responses during slow-wave sleep at frequencies of 6-8 c/sec and at flash intervals of 50 msec. The morphology of the discharges was similar to that seen in spontaneous discharges in deep sleep. The discharges, however, were stimulus-bound and there were no poststimulation spike-wave afterdischarges, which characterized wakefulness and REM sleep. The findings imply that, in deep sleep, while the cortical response to photic stimulation via the specific pathway system remains intact, the cortex appears to be functionally disconnected from those subcortical mechanisms essential for afterdischarge perpetuation.


Lateral geniculate multiple-unit activity related to metrazol potentiated after-discharges. Following parenteral administration of subconvulsive levels of pentylenetetrazol (Metrazol) photic stimulation induced an augmented rhythmic sequence of late neuron population burst-inhibition periods in the dorsal lateral geniculate nucleus (LGN) of the rat. This late bursting-inhibition activity was in turn observed to be associated with the augmentation of cortically recorded photically evoked after-discharges (PhADs). Multiple-unit activity (MUA) was also recorded from superior colliculus (SC), reticular formation (RF), posterior thalamic area (PTN), and dorsal hippocampus (HIPP). Only SC and RF exhibited an initial discharge to photic stimulation with late bursting infrequently observed and only in the SC. PTN showed some tonic increases in MUA following photic stimulation. HIPP MUA was essentially unaffected by photic pulse stimulation. The results document the neuronal role of the LGN in PhAD activity and were discussed in terms of a recurrent LGN inhibitory system governing cortical PhAD production and elaboration.


Suppression of cortical epileptiform activity by generalized and localized ECoG desynchronization. The effects of high frequency electrical stimulation of both diffusely projecting brain regions and regions of more restricted projection were studied on penicillin-induced cortical epileptiform focal activity in the cat. Results obtained were contingent on the level of focal activity present at the time of stimulation. Very active foci (spike rates above 0.5/sec) were uniformly driven by stimulation of all structures under study. Foci exhibiting weak to moderate levels of activity were, on the other hand, inhibited both during and following stimulation. Episodes of spike suppression induced through stimulation of diffusely projecting structures were typically followed by an intensified "rebound" of interictal activity. Episodes of suppression induced through stimulation of regions of limited projection were not followed by such rebounds, an effect most dramatically apparent with caudate stimulation and motor cortex foci. Results are discussed in terms of the interaction between naturally occurring brain rhythms in sleep and arousal with the epileptic process.


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.


Stabilization of alpha frequency by sinusoidally modulated light. Two experiments demonstrate that a spontaneous electroencephalographic (EEG) rhythm, alpha, can be frequency-stabilized by sinusoidally modulated light (SML) but not by photic flash stimulation. It is suggested that this frequency stabilizing effect is not due to superimposition of an evoked response on the background EEG. Instead, SML might phase-lock the scalp-recorded alpha rhythm by influencing subcortical alpha generators within narrow limits set by the inherent resonant EEG frequency.


EEG responses to photic stimulation in persons experienced at meditation. The EEG responses to intermittent photic stimulation were examined in a group of subjects experienced in meditation, and compared with those of a control group. The meditators exhibited a significantly smaller decrement in alpha activity and alpha blocking over the course of the experiment than did the control group, and alpha induction occurred earlier and more frequently in the meditators. These findings support the hypothesis that experienced meditators spontaneously enter the meditative state on closing the eyes, and also the view that physiologically the meditative state is one of prolonged drowsiness. An alternative interpretation, that meditation is a state of sustained attention, is discussed.


The click-evoked post-auricular myogenic response in normal subjects. The presence of the posterior auricular myogenic reflex was infectigated in healthy subjects. Click stimuli were delivered alternately to each ear and both ipsilateral and contralateral responses were recorded simultaneously. Smiling and head down position increased the number of responses. With this method, in contrast to previous findings, it was possible to demonstrate bilateral auditory reception in 89% of the 45 subjects studied.


An on-line transformation of EEG scalp potentials into orthogonal source derivations. A new type of EEG derivation has been investigated. This derivation, constituting a practical implementation of the Laplace operator, detects source activity as it appears at the surface level of the scalp. It is realized in the 10-20 system of electrode placement basically as an analogue superposition of four bipolar derivations, forming a star-like configuration around each electrode. Visual estimation of the topographical origins of a pattern, is thus replaced by a more efficient on-line process, which derives the source activity at the position of each individual electrode. Practical correlation tests have shown that the separation of adjacent derivations is improved by a factor of between two and four, compared to the corresponding bipolar and common reference derivations. Any feature of local origin will therefore have a correspondingly increased signal-to-noise ratio prior to the stage of visual or automatic interpretation. As a consequence of the partition of the scalp field into 19 source zreas, instead of utilizing an arbitrary number of potential differences, one fixed montage with 19 recorder channels is sufficient to present the total surface activity, within the limits of resolution of the electrode system.


Changes in chick oviduct chromatin antigenicity following estrogen withdrawal. A microcomplement fixation assay was used to study changes in the antigenicity of chick oviduct chromatin following estrogen withdrawal. Our findings indicate that following estrogen withdrawal, chromatin from previously stimulated oviducts lost those antigenic determinants which were characteristic of the stimulated state. Withdrawal did not, however, result in the reappearance of antigenic sites characteristic of the unstimulated oviduct. These results suggest that estrogen stimulation of the immature chick results in an irreversible alteration of the oviduct chromatin.


Character of the stimulatory response of uridine kinase in rat livers to cycloheximide treatment. Administration of cycloheximde to adult rats resulted in the enhancement of uridine kinase activity in the liver. The increase of enzyme activity was not affected by adrenal secretion or uptake of food. Actinomycin D, hydroxyurea and puromycin injected simultaneously with cycloheximide depressed the increase of enzyme activity. On the contrary, administration of 5-azacytidine following cycloheximide resulted in further enhancement of uridine kinase. While the half-time of template RNA for uridine kinase in control rat livers was 24 h, for cycloheximide-stimulated enzyme it was about 11 h. At higher dose levels cycloheximide leads to an increased incorporation of both uridine and orotic acid into liver RNA; however, this effect was not observed in the starved animals. The mechanism of action of cycloheximide is briefly mentioned.


On the nature of the external surface of cultured human embryo fibroblasts: an ultrastructural and cytochemical analysis utilising stain and lectin probes. Surfaces of human embryo fibroblasts in vitro were reacted with stain and lectin probes for carbohydrate moeities either after or in the absence of treatment with low concentrations of surface-active enzymes and EDTA. Only testicular hyaluronidase significantly suppressed affinity-binding of all three agents, suggesting that acidic glycosaminoglycans are principle components of the cell's exterior.


A mouse hepatoma cell line which secretes several serum proteins including albumin and alpha-foetoprotein. A permanent cell line (BW) was established from a transplantable mouse hepatoma, BW7756, which produces alpha-foetoprotein (AFP). Three clones were isolated from the uncloned culture: BW1, BW2 and BWTG3. The cells of the latter clone, which was isolated after selection in the presence of thioguanine, are deficient in the enzyme hypoxanthine-guanine-phosphoribosyl transferase. Both BW1 and BWTG3 cells have mean chromosome number of 64 (60 telocentric and 4 metacentric chromosomes). All three clones secrete at least five serum proteins into the culture medium: albumin, AFP, and alpha 2 globulin, transferrin and C3, the third component of complement. The approximate rate of albumin secretion by BW1 and BWTG3 cells is 10 mug/24 h/10(6) cells. Both albumin and AFP can easily be detected in cell extracts. The simultaneous production of AFP and a hepatocyte specific marker (albumin) by cloned hepatoma cells show that the production of AFP by the tumour is due to the tumoural hepatocytes themselves.


Effect of antitumor agents on sarcoma-180 tumor cells transplanted to liver, kidney, and lung. The survival time of animals, inhibition of the incorporation of thymidine-[6-3H] (3H-TdR) into DNA, and histopathological observation were made after the injection of Mitomycin-C, Bleomycin, cyclophosphamide, Daunomycin, Actinomycin-D, or 5-fluorouracil into mice transplanted with sarcoma-180 to their liver, kidney, and lung. The most prolonged survival time was obtained by the injection with cyclophosphamide and a moderate prolonged survival by Bleomycin and Actinomycin-D. In the case of 5-fluorouracil and Daunomycin, there were extreme variations in the survival time depending on the site of tumor growth. Cyclophosphamide and 5-fluorouracil showed greater and longer lasting inhibition of the incorporation of 3H-TdR into DNA of the tumor tissue, whereas the remaining agents caused transient inhibition on the tumor tissue. Inhibitory ratio and duration of the incorporation of 3H-TdR into DNA of normal site of the tissue of tumor-bearing organ were found to be more increased or almost the same compared with those of the tumor tissue. The most rapid recovery of the incorporation of 3H-TdR into DNA was observed in the small intestine among various organs and tumor in any treatment groups. From the histopathological observation, the degree of tumor cell damage by the agent was almost in agreement with inhibition of the incorporation of 3H-TdR up to 72 hr after the treatment.


Establishment and some biological characteristics of human hepatoma cell lines. Two cell lines of human hepatoma, HLE and HLF lines, were established in vitro from the hepatocellular carcinoma of a 68-year-old patient. One clone (HLEC) was obtained from a single HLE cell. The cells of HLE and HLEC were epithelial-like and both of these cells demonstrated glycogen granules in the cytoplasm when stained with periodic acid and Schiff reagent. Although HLF cells resembled fibroblasts in morphology, they appear to have originated from hepatoma cells, judging from epithelial characteristics in aggregates reconstituted by rotation culture and heterotransplantability. HLE cells produced alpha-fetoprotein until day 187 of culture, but HLF cells did not produce alpa-fetoprotein at any period examined. Chromosome number of both cell lines was distributed near the triploid range. HLF cells were transplantable into the cheek pouch of adult hamsters treated with cortisone acetate, but not HLE cells.


Isolation of parietal cells from guinea-pig gastric mucosa and the immunological characterization of their antigenic structure. A method is described for the isolation of parietal cells from the gastric mucosa of the guinea pig by enzymatic digestion with collagenase. A suspension was obtained that contained 70-80% parietal cells. About 80% of the cells were viable immediately after incubation, but viability dropped sharply after one hour. Parietal cells were identified by their morphology on light and electron microscopy, by their uptake of neutral red, by immunofluorescent staining and by carbonic anhydrase activity. Antibodies to four distinct parietal-cell antigens were obtained from rabbits immunized with the isolated parietal cells or fractions thereof. These antibodies were directed against the microsomal fraction of the parietal-cell cytoplasm, the plasma and nuclear membranes, the soluble proteins, and Castle's intrinsic factor. The antibody against the microsomal fraction, though reacting in the same way as the antibody to parietal cell canaliculi found in the serum of patients with pernicious anaemia, showed greater species specificity.


[Cytochemistry of leukocytes in childhood. III. Changes of cytochemical reactions in lymphocytes in infections and during immunological reactions]. The present paper gives a report on changes of enzyme reactions, of the glycogen content, and the nucleolus picture in lymphocytes which are based on cytochemical investigations of blood smears taken from 110 children with different diseases. In 20 new-born babies the cytochemical responses of lymphocytes after triple immunization with Di-Pe-Te immunization matter were observed. The findings reveal significant changes to be found predominantly in the activity of alpha-naphthylacetate esterase, PAS-reaction and the nucleolus picture of lymphocytes in immunological reactions. No hints for specific immunological functions of lymphocytes could be detected. The changes may refer to B-lymphocytes and to T-lymphocytes.


Neutrophil alkaline phosphatase activity in the early puerperium. Neutrophil alkaline phosphatase studied cytochemically according to Kaplow's method in 20 healthy women aged 20 to 38 years, shows statistically significant, although transient, increase on the second day after physiological delivery and returns to initial values after several days. The above increase is unrelated to the increased enzyme activity observed in the terminal months of pregnancy in a majority of healthy women. It is associated with an increase in neutrophilic granulocytosis in the puerperial period.


[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.


The use of 75selenomethionine for labelling the formed and protein elements of the blood. In the introduction the authors describe the biological role of platelets, plasma protein, fibrinogen and erthrocytes as well as the methods of tagging, separating and measuring them. The advantage of platelet labelling with 75selenomethionine is that it allows production and life-span to be determined simultaneously, however, the in vivo measurements do not give information on the site of platelet sequestration. Protein anabolism as well as katabolism can be evaluated with it, and the simultaneous determination of platelet and fibrinogen turnover may be especially useful in pathological conditions. The determination of erythrocyte life-span with 75 selenomethionine does not give so much information as the 59Fe and 51Cr methods, but when used for the examination of cell kinetics in combination with the latter methods it may afford an insight into erythropoiesis. The biological and the effective half-life periods (Teff) of 75selenomethionine are probably shorter than indicated in the literature.


[Studies on the osmotic resistance and the viability of amidinated erythrocytes]. The present studies are concerned with properties of amidinated erythrocytes. The reactions of dimethyladipimidate with proteins in solution and red blood cells, respectively, result in an intermolecular cross-linking. Following an amidination of human serum albumin or human gamma-globulin cross-linked products of increased molecular weight have been demonstrated by polyacrylamide gel and immune electrophoresis. Human erythrocytes previously amidinated intensely, exhibit a restricted motility of membrane particles and cross-linked hemoglobin. Intensely amidinated erythrocytes are resistant against distilled water, and they do no longer agglutinate. The findings presumably indicate an increased permeability of the amidinated red cell membrane. The glycolytic activity was found to be normal in moderately amidinated erythrocytes. In comparison with normal red blood cells, previously moderately amidinated erythrocytes of the rat become sequestered more quickly after re-injection into the vascular system.


Frozen-thawed red cells--standard article for hospital use? The freezing and storing of glycerol protected red cells allows the present 20---40 day limitation of red cell storage to be indefinitely lengthened. Although frozen thawed cells are more expensive to prepare than ordinary packed cells, the longer shelf-life of the red cells, the reduced risk of hepatitis transmission, and the reduction of immunization against leuko- and thrombocytes, are all advantages which out-weighed this additional cost. To effect an increase in their use, frozen thawed cells could be distributed from central blood banks to hospitals were they would be stored until needed for transfusion. Thawing and washing would necessarily be done by the hospital blood bank personnel.


[Further improvement of the ACD-AG protective solution for blood. II. Behavior of the oxygen affinity of preserved blood in ACD-AG medium and in a protective solution with addition of pyruvate and xylitol and elevated Ph values]. The present paper deals with the behaviour of the oxygen transport function as well as the 2.3 DPG and ATP levels of erythrocytes during the storage in an ACD-AG solution. In the ACD-AG blood in P 50 fell from 20mm of Hg to values of 12 mm of Hg within 4 weeks of storage. The 2.3 DPG content had already fallen to values below 10% within a fortnight. Additions of xylitol (10 mM in the blood) and pyruvate (0.3 mM in the blood) will delay the decrease of P 50 and the 2.3 DPG content. Concerning the ATP behaviour there was no significant difference between ACD-AG blood and that with additions of xylitol and pyruvate. Up to a storage of a fortnight stored blood in the ACD-AG solution of xylitol and pyruvate will be equal to ACD-AG fresh blood as far as the parameter of oxygen transport function is concerned.


[Further improvement of the ACD-AG protective solution for blood. III. Improvement of the oxygen transport function of erythrocytes in sorbitol xylitol pyruvate solutions with elevated Ph]. If the pH value in the ACD or in the ACD-AG storage solution is enhanced, the glucose in the autoclaving with undergo a caramelizing process. For this reason glucose was replaced by sorbite in the storage solutions with a pH value of 6.0 and additions of xylitol and pyruvate. The initial pH value in the blood amounted to 7.3. The content of 2.3 DPG of the erythrocytes remained fully preserved in the blood with sorbitol and additions of xylitol and pyruvate during the first 2 weeks of storage and decreased to 30% only in the third week. There were only slight amounts of 2.3 DPG in the ACD-AG blood at that time of storage. Up to the third week of storage the ATP content of erythrocytes as well as the haemoglobin level in the plasma revealed no essential differences between stored blood with sorbitol and xylitol as a substrate or glucose + xylitol respectively. The quick decrease of the ATP level to zero and the simultaneous strong increase of haemolysis in the sorbitol blood within the fourth week of storage is discussed in connection with a lowering of the NAD/NADH2 quotient. For the purpose of keeping the 2.3 DPG level of erythrocytes a storage solution with sorbite and xylitol (ASCX-AG-Pyr 10mM) seems to be well suited for a storing time of 2---3 weeks at first.


A new abnormality of platelet functions. Association of storage pool disease (thrombocytopathia A) with impaired reactivity of platelets to collagen. Multiple platelet abnormalities were found in a patient with bleeding symptoms. The platelet content of ADP and PF 4 was decreased and the uptake of 14C-serotonin was impaired. The content of acid phosphatase, beta-glucuronidase and beta-N-acetylglucosaminidase was, however, normal and these enzymes were normally released or made available by bovine fibrinogen or ADP. There was no adhesion of platelet to collagen, which also failed to induce reptilase clot retraction, platelet aggregation and release of any of the platelet constituents. The platelets therefore exhibited signs of thrombocytopathy of a combined type with a decreased storage pool as well as a qualitative dysfunction with impaired reactivity to collagen.


[Methodological studies on the immunologic determination of proconvertin (factor VII)]. The influence of the different determinations of factor VII activities on the determination of the concentrations of the immunoreactive factor VII in the neutralizing test according to Good-Night were re-examined. The measurement of activity was performed with Seitz filtered cattle plasma as a complex proof of factor VII and X and with a specific artificial deficiency plasma. The examinations had the following results: In the plasma of healthy grown-up people there is a considerable, apparently physiologic range of dispersion of the immunoreactive concentration of factor VII within its normal activity which can be traced irrespective of the used measurements of activity. In plasma samples with a decreased activity of factor VII the values of measurement of the complex evidence will surpass those of the specific determination. The extent of neutralizing the activity in factor VII is not only dependent on the antibody concentration, but will mostly depend on the antibody-antigen relation. The findings obtained prove that the natural deficiency plasma of factor VII used in the original method of Goodnight can be replaced by an artificial substrate plasma.


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.


Sequential Q- and Acridine orange-marker technique. A standardized Q- and acridine orange (AO)-fluorescence dual marker technique was described. It involved preservation of unstained chromosome slides in a vacuum desiccator up to 18 months, Q-staining, destaining, and treatment in Hanks' solution, pH 5.1, at 85 degrees C for 13 min, and acridine orange staining. Q-markers were found at the paracentromeric regions of chromosomes 3 and 4, the short arms and the satellites of the acrocentric chromosomes, while AO-marker spots were on the satellite-stalks of the acrocentrics. The advantage of the dual marker technique was illustrated by the determination of the origin of trisomy 22 in a spontaneous abortus.


Variation in human acrocentric chromosomes with acridine orange reverse banding. Twenty-five normal subjects were studied by acridine orange reverse (RFA) banding in order to obtain a preliminary estimate of the type and frequency of variations in color and length. Color variations were classified into 1 of 6 colors and size variations into 1 of 5 levels. The same cells were also studied by Q banding. Acridine orange reverse banding was found to be more useful than Q banding for characterizing variations in chromosomes 14, 15, 21 and 22. In addition, it was found that there was no consistent relationship between pale or bright Q banding and the various colors observed with RFA banding. For the optimal characterization of a chromosomal variation, multiple banding technics, including RFA banding, are necessary.


[Immunosuppression mediated by antibodies (author's transl)]. Until quite recently, it was generally believed that antibody-mediated immunosuppression is purely effected by virtue of covering up antigenic determinants in the periphery of the immune system, thus preventing any contact of the antigen with immunologically competent cells within the central parts of the immune system. But this conception was not able to give a plausible explanation of experimental data obtained during the last years. It was therefore repeatedly postulated that the passively administered 7S antibody may have a "central" effect, whereby both B- and T-cells were considered as target cells. The experimental data available are discussed in connection with the possible consequences for the administration of antibodies in man.


[Nature, character, occurrence, and demonstration of hepatitis B antigens (author's transl)]. The morphological, chemical and physical properties of HBAg suggest that the 42 nm component of the antigen, the Dane particle, represents the agent of viral hepatitis B. Its core contains a circular, double stranded DNA, a DNA polymerase and carried HBc-Ag. HBc-Ag is localized on the 21 nm particle, the tubular structures and the surface of the Dane particles. At least 8 different subdeterminants of HBs-Ag could be distinguished by means of specific animal anti-sera. HBs-Ag activity was demonstrated in almost all body fluids and excreta. The results of combined histologic, fluorescent and electronmicroscopic studies suggest ath HBc-Ag is localized in the liver cell nucleus and that HBs-Ag is found in the cysterna of the smooth endoplasmatic reticulum of the hepatocytes. The demonstration of HBs-Ag and the specific DNA polymerase in the serum indicate a hepatitis b virus infection with persistent reproduction of the agent, while demonstration of anti-HBs indicates that the infection has been overcome. The clinical importance importance of anti-HBc is controversial.


[Morphological characteristics and pattern of hepatitis B antigen expression in liver tissue in acute and chronic forms of hepatitis (author's transl)]. Immunofluorescent and electron microscopic methods allow the localization of hepatitis virus B-components at the cytologic and histologic level. The acute and chronic forms of viral hepatitis are re-evaluated in the light of these new findings. In HB-Ag-positive hepatitis four types of correlation between the expression pattern of HG-Ag components and different forms of hepatitis are recognized which are of diagnostic and prognostic relevance (elimination type, immunosuppression type, aggression type, and carrier type). The correlation between antigen expression, persistence of infection and type of inflammation seems to reflect the specific immune state towards the hepatitis B virus.


Induction of allergic encephalomyelitis using hydrosoluble adjuvant and the tryptophan region of myelin basic protein. Experimental allergic encephalomyelitis has been induced in guinea pigs using the encephalitogenic tryptophan peptide as antigen and a hydrosoluble adjuvant extracted from Mycobacterium tuberculosis, var. hominis, strain H37Ra. The maximum response was observed using 100mug of adjuvant per animal. This is a quantity of adjuvant substantially higher than was necessary to induce disease utilizing the whole myelin basic protein as antigen.


Contact sensitivity in mice measured with thymidine labeled lymphocytes. An ear assay was used to detect delayed responses in mice. Animals were sensitized and subsequently injected with 3H thyminedeoxyryribose to label antigen sensitive cells. Each animal was given a control and a test ear challenge and the respective responses were assessed by scintillation counting of 3H in cells which had accumulated at the reaction site. Delayed responses were consistently observed in animals sensitized to dinitrochlorobenzene (DNCB) but not in animals sensitized to sheep red blood cells. Reactivity to DNCB was minimal in tolerant animals and animals sensitized to the control agent. Both peritoneal exudate and spleen cells of antigen-sensitized animals were found to contain more label than cells from non-sensitized animals.


Antigen-induced inhibition of experimental allergic encephalomyelitis. IV. Studies of the C-terminal end of the myelin basic protein molecule (1). The C-terminal end of the myelin basic protein (BP) molecule (peptide 117.170) was obtained by cleavage with BNPS-skatole, and purified by gel filtration. Peptide 117-170 from guinea pig BP induced experimental allergic encephalomyelitis (EAE) in Lewis rats, whereas the corresponding peptide from bovine BP was inactive. Neither peptide showed more than trace activity when tested in guinea pigs. Guinea pigs and rats treated with peptide 117-170 in incomplete Freund adjuvant were not rendered unresponsive to EAE induced by subsequent challenge with an encephalitogenic emulsion of BP in complete Freund adjuvant. Indeed, peptide-pretreated rats tended to develop clinical EAE significantly earlier than untreated controls.


Influence of molecular structure on the tolerogenicity of bacterial dextrans. I. The alpha1--6-linked epitope of dextran B512. Native dextran B512 is a near-linear glucose polymer with 96 per cent alpha1--6 and 4 per cent alpha1--3 linkages and a molecular weight (mol. wt) of 8 X 10(7). Sheep RBC sensitized with its O-stearoyl derivative (prepared by a modified method) have been used satisfactorily in direct PFC assays. B512 immunizes BALB/c mice optimally with doses of 1--10 mug and produces B-cell tolerance with 1 mg upwards. The specificity of the response determined by PFC inhibition analysis, is directed towards an alpha1--6-linked epitope. High dose tolerance is not preceded by immunity and is stable on cell transfer to irradiated recipients in which responsiveness becomes perceptible after 4--6 weeks. Progressive depolymerization of this polysaccharide reduces immunogenicity and tolerogenicity, both of which are extinguished when the mol. wt falls to 2 X 10(4). Optimal immunization with B512 is succeeded by partial tolerance (previously characterized by analogous levan experiments as a B-cell exhaustion process). The tolerance threshold dose of B512 is reduced 1000-fold during immunosuppression with cyclophosphamide. PFC inhibition studies supported the contention that tolerogenicity of polysaccharides is influenced by their overall binding capacities. A direct relationship between inhibitory and tolerogenic activities was found both with B512 fractions of varying mol. wt and with heterologous dextrans. The similarities between B512 and levan argue against the association of a highly branched structure with greater tolerogenicity. The effect of reducing the percentage of alpha1--6 linkages in dextrans suggests, however, that epitope density probably plays a contributory role in determining the outcome of interaction between polysaccharides and B cells.


Influence of molecular structure on the tolerogenicity of bacterial dextrans. II. The alpha1--3-linked epitope of dextran B1355. Dextran B1355 is a branched glucose polymer containing 57 per cent alpha1--6, 35 per cent alpha1--3 and 8 per cent alpha1--2/1--4 linkages. Direct PFC responses to B1355 can be measured with sheep RBC sensitized with its O-stearoyl or palmitoyl derivative, and, as shown by inhibition analysis, are specific for an eptiope which is dependent on alpha1--3 linkages. B1355 is a potent immunogen in BALB/c mice producing peak PFC levels which approach 10(6) per spleen following an optimal dose of 1 mg. By contrast, the alpha-1--3-linked epitope of B1355 is feebly tolerogenic, for even 10 mg still induces a strong initial response. Mice given 1--10 mg sustain PFC levels 1--2 log10 above background for several months, but do not respond further to restimulation. Full recovery is attained by their spleen cells within 1 week of transfer into irradiated recipients. Deeper tolerance to this epitope was attained in vivo only when these larger doses of B1355 were injected during cyclophosphamide suppression. Two exceptions to this weak tolerogenicty were found. First, BALB/c spleen cells developed durable partial alpha1--3 tolerance following 2-hour incubation with B1355 in vitro. Second, CBA mice were fully tolerized by doses of 1 mg upwards. It is argued from these and other data in the accompanying papers that the relative resistance of BALB/c mice to induction of alpha1--3 tolerance is explicable neither as part of a more general phenomenon based on macrophage activity nor as due to an inadequate epitope density. A possible explanation based on features of the genetically determined high alpha1--3 responsiveness of BALB/c B cells is discussed.


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'.


Specificity of the antibody response in inbred mice to bovine type I and type II collagen. Mouse antibodies to soluble bovine skin (type I) collagen react with determinants which are located in the rigid triple-helical portion of the antigen and become destroyed upon unfolding the molecule. Helical antigenic determinants are dependent on the genuine chain assembly, e.g. alpha[1(I)]2alpha2. Artefactual triplehelical structures of the composition [alpha1(I)]3 or [alpha2]3 or a genetically distinct type II collagen from cartilage showed no or only weak cross-reactivity. Pepsin treatment of type I collagen known to remove short, non-helical sequences at both ends of the molecule had virtually no effect on antigenicity and immunogenic activity. A radioimmunoassay failed to detect antibodies in three congenic resistant mouse strains immunized with denatured type I collagen. These strains had been previously classified as high or low responders to native type I collagen. Agglutination titres vs denatured collagen culd already be demonstrated in nonimmune sera. The agglutinating activity was labile against heating at 56 degrees and could not be increased by immunization. Two out of five inbred strains showed a high response against pepsin-dissolved bovine type II collagen with the chain composition [alpha1(II)]3. Lack of correlation in the responder state to both collagen types indicated control by different immune response genes. Antibodies to type II collagen also reacted against triple-helical antigenic determinants and showed neglible cross-reaction with type I collagen.


Studies on the control of antibody synthesis. VII. Change in affinity of direct and indirect plaque-forming cells with time after immunization in the mouse: loss of high affinity plaques late after immunization. The change in avidity of anti-hapten antibody with time after immunization was studied in mice at the level of the antibody-forming cell. A progressive increase in avidity was seen in both direct and indirect plaque-forming cells. Late (38 days) after immunization with a large dose of antigen there was a preferential loss of high avidity plaque-forming cells and the average avidity decreased. High avidity memory cells were still present since boosting resulted in the prompt appearance of very high avidity plaque-forming cells. There was a strong positive correlation between the avidity of the direct and the indirect plaque-forming cells present in the same spleen. A pattern of change in avidity and heterogeneity of avidity similar to that observed with intact animals was seen in lethally irradiated mice reconstituted with normal spleen and thymus cells.


The potentiality of antibody-producing cells. II. Evidence for two antibody molecules of different specificities secreted by micromanipulated bispecific mouse spleen cells. Bispecific PFC appearing on the 4th day after immunization of mice with sheep erythrocytes conjugated with trinitrophenyl hapten (TNP-SRBC) were identified by their capacity to lyse native SRBC and TNP-conjugated horse erythrocytes (TNP--HoRBC) simultaneously in an open carboxymethylcellulose medium. Individual PFC thus detected, were micromanipulated into two successive media containing the indicators SRBC and TNP--HoRBC. Out of 103 transferred double cells ninety-two (89 per cent) remained double in the second medium and of the ninety-two transferred into the third medium, fifty-four (58 per cent) provoked a double lysis, eighteen (19 per cent) provoked a single lysis and twenty (23 per cent) ceased to lyse. On the other hand, when the second medium contained the indicators and the soluble specific inhibitor (TNP--BSA), out of 156 transferred double PFC, 125 (80 per cent) became single and only nineteen (12 per cent) remained double. Of these cells, 136 were transferred into the third medium (not containing inhibitor) and here again sixty-five (48 per cent) became double, forty-nine (36 per cent) remained single and twenty-two (17 per cent) ceased to provoke any lysis. Other double PFC were micromanipulated from the original revealing medium into two successive media containing only one indicator and the homologous (second medium) or the heterologous third medium) soluble inhibitor (TNP--BSA or soluble SRBC antigen), in order to see whether a soluble inhibitor suppresses only the corresponding specific lysis. Out of 186 double PFC transferred into the media containing the indicator and the corresponding inhibitor 147 (79 per cent) were specifically inhibited, whereas out of 176 double PFC transferred into the third medium (in which about 20 per cent of the cells cease to function) containing one indicator and the unrelated inhibitor, 117 (66-6 per cent) lysed the indicator in spite of the presence of the unrelated inhibitor. Since a specific inhibitor suppresses the lysis of the corresponding indicator, whereas its presence does not interfere with the lysis of the unrelated indicator in the majority of double PFC, it can be concluded that these cells secrete two types of antibody molecules possessing different specificities.


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.


[Long term tolerance of highly dosed infusions of hydroxylethylstarch and dextran 60 in normovolemic rabbits]. Normovolemic rabbits were administered 50 ml of 6% hydroxyethyl starch in saline, 6% dextran 60 in saline solutions 5 days a week over a period of 3 weeks. Only 2 rabbits of the hydroxyethyl starch or the dextran treated series and 4 rabbits of the saline series survived 3 weeks. In all 3 series large amounts of fluid in the serous cavities and edema of the lungs due to the overload of fluid have been observed. Severe hydropic changes were found in the liver and kidneys of the hydroxyethyl starch and dextran treated series, but not after prolonged administration of saline alone. According to the findings presented there is no difference between the effects due to the prolonged administration of extremely high doses of hydroxyethyl starch and dextran 60.


The Duffy blood group system: a review of recent developments. Five antigenic specificities are associated with the Duffy blood group system. Fya, Fyb, By3 and Fy4 are under the genetic control of a locus near the centromere of chromosome 1. Fy5 is absent from Fy (a-b-) red cells and also from cells that have the Rhnull phenotype. It appears that interaction between Duffy and Rh gene products may be necessary for synthesis of Fy5 antigen. Most Caucasians have a Fya or Fyb gene (or both), inherited by strict Mendelian principles. No evidence has been found to suggest that independently segregating Duffy modifying genes exist. The frequency of Duffy genes varies greatly in different sections of the world population. Aboriginal populations have a high frequency of Fya, Fyb is the most common gene in European Caucasians, and Negroes have a high frequency of an allele named Fy. Anti-Fya is the most common Duffy antibody and is found most frequently in the serum of Caucasians immunized by blood transfusion. The Fya antigen is about 40 times less immunogenic than K antigen of the Kell system. Only about one anti-Fyb serum is found foe every 20 that are anti-Fya. The Fya and Fyb red cell antigen receptors are inactivated by proteolytic enzymes but not by neuraminidase. Sialic acid does not appear to be an essential component of the antigen structure. Duffy antigens appear to be thermolabile membrane proteins and they are not present in a soluble form in plasma or in body secretions.


[Serological identification of carcinospecific antigens (and their significance as donor screening or for specific groups of diseases)]. Alpha-1-fetoprotein is an example of a circulating, measurable tumor product of diagnostic and therapeutic value. The involvement of  its synthesis could be a result of a premalign cellular change in cell biochemistry. Contrary to the synthesis of trophic hormones in certain undifferentiated neoplasms, alpha-1-fetoprotein in hepatoma is specific for the organ origin of the tumor. Unlike the immunoglobulins in myeloma or the corticosteroids in adrenocortical tumors, it is a protein that normally can be synthetized in the fetus only. The purpose of the present paper is to discuss a new method for testing serum samples of blood donors being suspected of an alpha-1-protein by means of counterelectrophoresis. The diagnostic value is shown in a blood donor who could be singled out as a suspect of primary liver carcinoma only by means of serological testing.


Immune complexes in the spleen. Replacement of immune complexes trapped in spleen follicles by new immune complexes from the circulation. The fate of intravenously injected 125I-BGG-anti-BGG in the spleen of mice was studied using autoradiography. Part of the labelled immune complexes was trapped in the follicles of the spleen as could be expected. In a first experiment it was found that injections with unlabelled immune complexes were followed by a partial release of the labelled immune complexes from the follicles. In a second experiment unlabelled immune complexes retained in spleen follicles appeared to inhibit the trapping of intravenously injected labelled immune complexes to some degree and for some time. The conclusion was drawn from these experiments that immune complexes, which normally remain in part of the lymphoid follicles for a long period, may be replaced by new immune complexes from the circulation. This seems important since trapping in lymphoid follicles of antigen complexed by antibody is the only known mechanism by which small amounts of antigen may be preserved in the body for a long time after the initiation of antibody production. The bulk of antigen and antigen-antibody complexes is removed by phagocytosis followed by destruction. It appeared also that, although all spleen follicles in the mouse spleen is able to retain the complexes for a longer time. Possible explanations for these individual differences between the follicles of one spleen are discussed.


Influence of surface hapten density of sheep red blood cells on the hemolytic or blocking activity of IgG antibodies. The hapten density on the surface of sheep red cells, coated with benzylpenicilloic acid (BPO) was determined. This was achieved by comparison of the inhibitory capacity of the test cells with that of a standard cell preparation of known antigen density. IgG-induced hemolysis was much more affected by the recution of haptenic sites/cell than IgM-induced hemolysis. It was concluded that more than 18,000 protein islets on the sheep red cell surface bearing at least four haptenic sites were necessary for IgG-induced lysis. Target cells with 190,000 BPO groups/cell could be protected by anti-BPO-IgG against the lytic effect of simultaneously added IgM. Using target cells with 240,000 haptenic groups/cell or more a synergistic hemolytic action of IgG-and IgM-anti-BPO antibodies could be observed.


Differential histamine release by dextran and the ionophore A23187: the actions of inhibitors. Inhibitors of mast cell membrane activation reduced histamine release from rat mast cells induced by dextran and phosphatidyl serine but not that induced by the calcium ionophore A23187. Such inhibitors included cromoglycate, an orally-active anti-allergic agent 3-(5-tetrazolyl)thioxanthone 10,10-dioxide, dibutyryl cyclic 3':5'-AMP, theophylline and dicumarol. Inhibitors of mast cell metabolism reduced both types of release and these included oligomycin, papevevime, and the two uncouplers of oxidative phosphorylation-alpha2,4-dinitrophenol and CCCP. Inhibition of histamine release from rat isolated peritoneal mast cells by either a mixture of dextran and phosphatidyl serine or the ionophore A23187 thus allows inhibitors of mast cell membrane activation to be distinguished from those affecting cell metabolism or the later stages of the secretory process.


Studies on IgE in human intestinal fluids. A fragment of IgE with molecular weight of about 40,000 was identified by radioimmunoassay in human small intestinal fluid after fractionation by gel filtration chromatography. Digestion of E myeloma protein PS by pooled intestinal fluid, trypsin or chymotrypsin yielded a degradation product of similar molecular weight that probably consisted principally of epsilon 1 determinant-containing fragments. These findings suggest that IgE is secreted into the intestinal lumen and degraded there by pancreatic proteolytic enzymes, producing an enzyme-resistant portion of the amino-terminal part of the Fc region.