Herpes simplex vaccines.

Herpes simplex vaccines. Prospects for the development of vaccines against herpes simplex virus types 1 and 2 and other herpesvirus group vaccines are discussed from the scientific, medical, and economic standpoints. Probably, the most practical development will be glycoprotein subunit vaccines, prepared from herpes simples virus types 1 and 2, that will be tested for prophylaxis and therapy of acute viral disease and for cancer that may be caused by these agents.


Combination chemotherapy of L1210 leukemia with 1-beta-D-arabinofuranosylcytosine and 5-azacytidine. 1-beta-D-Arabinofuranosylcytosine (cytarabine; ara-C) and 5-azacytidine (5-azaCR), cytosine nucleoside antimetabolites with different mechanisms of action, are both effective in the treatment of human leukemia, and the clinical use of these two agents in combination has been suggested. We have studied the therapeutic effect in L1210 leukemic mice of single i.p. doses of ara-C and 5-azaCR in combination. Therapeutic effects observed depended markedly on the sequence and time interval between the doses of each agent. Antagonism was observed when both agents were administered simultaneously. The optimal therapeutic effect was observed when 5-azaCR was administered after ara-C at a time when tumor DNA synthesis had maximally recovered after the ara-C dose. The dose-interval effect and correlation with recovery of DNA synthesis capacity were also observed in studies in vitro in which the survival of L1210 cells in culture was examined. ara-C was shown to inhibit the incorporation of [4-14C]-5-azaCR-derived radioactivity into DNA of L1210 cells in culture, and the therapeutic effects observed are interpreted in terms of these latter results and the mechanisms of action of the two agents.


Molecular interactions of the combined effects of bleomycin and x-rays on mammalian cell survival. The interactions between bleomycin and X-ray damage and repair have been examined in rat and human tumor cells. Bleomycin itself indices extensive DNA single-strand breaks but does not appear to inhibit the repair of X-ray-induced DNA single-strand breaks. Quantitative analysis of these interactions is complicated by the retention of active bleomycin within cells that remains capable of further DNA degradation even under the conditions of alkaline sucrose gradient cell lysis. DNA double-strand breaks and/or disruptions of DNA-lipid complexes also occur following bleomycin exposure. X-ray-induced excision repair replication is only minimally influenced by even high concentrations of bleomycin. A small amount of excision repair is demonstrable in nonirradiated cells treated with high concentrations of bleomycin consistent with repair of bleomycin-induced nucleotide damage in cellular DNA by a "cut and patch" repair mechanism. Repair of bleomycin-induced DNA single-strand breaks also occurs. The data indicate that bleomycin and X-ray damage are quite similar both in their induction and repair, but that lesions occur and are repaired independently. The enzymatic mechanisms appear similar in the two cell types despite substantial differences in their sensitivity to bleomycin.


Pulse cytophotometric analysis of cell cycle perturbation with bleomycin in vitro. The kinetic response of a human lymphoma cell line to bleomycin has been analyzed, using an ICP-11 pulse cytophotometer. Bleomycin induced a delay of the cell-cycle traverse in G2 phase, the extent and recovery of which depended on drug concentration, exposure time, and cell cycle stage where treatment was applied. Different phase sensitivity for lethal damage (G2 phase) and kinetic response (early S phase) were documented. Recovery from G2 block did not predict for unimpaired reproductive capacity.


Microfluorometry of nuclear acridine orange metachromasia in lymphocytes of thymus, spleen, and blood of AKR and random-bred mice. Lymphocyte chromatin lability to acid hyrolysis was studied using acridine orange fluorescence metachromasia in a high-lymphocytic-leukemia-susceptibility strain (AKR) and random-bred mice (ICR). Comparisons were made of blood, thymus, and spleen lymphocytes between random-bred, "normal" AKR, and leukemic AKR animals. The leukemic mice were in the stages of the disease characterized by enlarged thymus and spleen but preceding massive elevation of blood lymphocytes. The ranges of the mean chromatin acid lability overlapped and were nearly identical in peripheral blood lymphocytes. However, thymic and splenic lymphocytes showed a marked rise in mean chromatic acid lability in the leukemic animals. The ranges of the mean values of this parameter were also found to be far greater in the lymphopoietic organs of normal AKR than in the random-bred mice. The data indicate that anatomically normal AKR animals of an age in which they are highly susceptible to spontaneous lymphocytic leukemia may contain a greater number of lymphoblasts in both the spleen and the thymus than do comparable random-bred mice. The implications of these findings are discussed in relation to strain differences and the concept of thymic origin of lymphocytic leukemia in mice.


Spatial distribution of synapses onto thoracic motor neurones in locusts. Aggregates of synaptic vesicles, stained black by the zinc iodideosmium procedure, can be visualised with the light microscope in 1 mum plastic sections. This allows the main branches of a neurone to be reconstructed relatively rapidly and the associated vesicle aggregates to be plotted. By resectioning, the identity of the vesicle aggregates has been confirmed with the electron microscope. Two flight motor neurones in the mesothoracic ganglion of the locust have been examined. One is identified as a dorsal longitudinal muscle motor neurone (muscle 112) and the other is probably a subalar neurone (muscle 99). Both have a large density of vesicle aggregates on the neuropilar segment, the widest part of the main neuronal axis, but few on the neurite within 250 mum of the cell body. The larger branches arising from the neuropilar segment tend to have a lower density of aggregates than fine branches, which suggests that synapses to the branches may occur mainly on the distal twigs. These results are an important preliminary step in determining the integrative functions of such neurons and have immediate implications in the interpretation of microelectrode recordings.


Quantitative changes of the C-cell population in the rat thyroid during postnatal ontogenesis. The number and distribution of C-cells in the rat thyroid gland, have been investigated during postnatal ontogenesis from birth to 120 days of age. The argyrophilic and metachromatic properties of these cells were used to identify them. In the thyroid of newborn rats the C-cells do not exhibit argyrophilia and metachromasia. These reactions appear at 10 days and can be seen at all subsequent ages. The number of C-cells shows a parallel increase with age as demonstrated by the change in the proportion of C-cells: F-cells: colloid: stroma during development. A marked increase in C-cells was found at 50 days of age when the proportion of C-cells rose to 27.67% from the value of 16.78% at 30 days. At 70 days a decrease was noted (20.50%) which hardly changed until 120 days of age (22.20%). The numerical increase in C-cells occurs at the expense of the follicular epithelium and stroma. The C-cells occupy elongated islet-like region in the central part of the lells are present. The long axis of the C-cells area is parallel with the longitudinal axis of the lobe. The area of C-cells is largest at the centre of the lobe, corresponding to the territory of the peak of the Gaussian curve for the numerical distribution of C-cells.


Comparison of the early chromatin reaction of the mouse thymocytes incubated with phytohemagglutinin and concanavalin A. The preliminary activation of mouse thymocytes in vitro following exposure to the mitogens phytohemagglutinin and Concanavalin A, was compared. For this purpose the ratio of the fluorescence intensity for acridine orange-DNA complex at light wave length gamma = 590 nm to the fluorescence intensity of this complex at light wave length gamma = 530 nm was determined. It was shown that 100% of thymocytes showed an increase in chromatin activity under the influence of Con A within 15 minutes of incubation, but that following incubation with PHA an increase in activity occurred only in 25% of cells and was detectable only after 60 minutes of incubation. The results show that differences in the reactivity of thymocytes to PHA and Con A concern not only the late steps of activation such as RNA, protein and DNA synthesis, but also involve the earliest responses to the mitogen. The observed differences are discussed and correlated with the different degrees of maturity of the thymocytes.


[Inhibition of antigenic competition by immunostimulants]. The diminution of immune response against SRBC induced in mice, by a prior injection of HRBC was counteracted by addition of certain immunostimulants to SRBC. The intensity of inhibition of antigenic competition was related to the quantity of immunostimulant added to SRBC. Some immunostimulants (B. abortus, lipopolysaccharide) were more active than others (C. parvum, Poly I : C). To inhibit antigenic competition immunostimulant had to be injected after or in mixture with SRBC never before.


[Molecular weight of human alpha2-H-ferroglobulin subunits. Comparison with molecular weight of ferritin subunits]. alpha2 H globulin, a glycoferroprotein, was first demonstrated in the sera of patients with malignant diseases. This protein was isolated from cancerous human liver, and compared with ferritin, a ferroprotein showing some identical properties (presence of iron, high molecular weight, common antigenic determinants). However, physicochemical differences were observed between these two proteins. The study of protein dissociation was performed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction by mercaptoethanol. A similar molecular weight of 19 000 is obtained for subunits of these two proteins. This value agrees well with the results obtained by other authors for ferritin.


Muscarinic effects of vagosympathetic trunk stimulation on the repetitive extrasystole (RE) threshold. The precise mechanism of the vagus in opposing adrenergic influences on cardiac vulnerability has not been defined. In the present investigation, cholinergic stimulation was produced by administering the selective muscarinic agent methacholine (MCh) and ventricular vulnerability was assessed by measuring the repetitive extrasystole (RE) threshold. MCh produced a sustained 98 +/- 12% (P less than 0.0001) increase in the RE threshold; the increase was abolished by concurrent infusion of atropine (0.2 mg/kg). When MCh was administered during beta-adrenergic blockade with propranolol, however, no further increase in RE threshold occurred beyond that resulting from propranolol alone. Infusion of norepinephrine (NE), in a dose insufficient to provoke systemic hypertension, significantly lowered the vulnerable period threshold. This decrease was completely abolished by concurrent vagosympathetic stimulation (VS). It is concluded that VS affects ventricular vulnerability through its muscarinic action and that the protection against VF is in part due to modulation of neural and humoral beta-adrenergic inputs.


E-type prostaglandins: a new emergency therapy for certain cyanotic congenital heart malformations. Prostaglandin E2 (PGE2) has been used to maintain patency of the ductus arteriosus in four neonates with cyanotic congenital heart disease due to obstructive right heart malformations. PGE2 was infused prior to surgery, and in three patients, during surgery until a satisfactory aorto-pulmonary shunt was established. PGE2 produced consistently an immediate and persistent rise in arterial oxygen saturation, which could be ascribed to dilation of the ductus arteriosus. No major side effects occurred, except for pyrexia in two infants. All patients recovered well from surgery. We propose this treatment as preparation for surgery in any infant with congenital heart defects and ductus-dependent pulmonary blood flow. The same treatment may be useful preoperatively in patients with aortic interruption who also depend on continued patency of the ductus for blood supply to the lower half of the body.


Immunofixation. I. General principles and application to agarose gel electrophoresis. Immunofixation offers the worker an economical means of physically locating a protein in an electrophoretic strip and is ideally suited to forensic medicine, genetic studies, or research. The method is as simple and economical as the commonly used one- or two-dimensional immunoelectrophoresis, yet yields considerably more information.


Cytochemical studies on RNP complexes produced by puff 2-48BC in Drosophila hydei: uranyl acetate and phosphotungstic acid staining. Differential staining of the core and RNP particles of RNP complexes in puff 2--48 BC in salivary gland chromosomes of Drosophila hydei was achieved with aqueous uranyl acetate (UA) at low pH, with UA in acetone, with phosphotungstic acid (PTA) in organic solvents, and with aqueous PTA at pH 5 And 6. A comparison of the results of UA and PTA staining under various conditions indicate that the proteins in the core region and in the RNP particles connected to it differ with respect to their amino-acid composition (arginine and lysine residues).--The staining mechanism of PTA and UA is discussed.


Human nucleolar organizer chromosomes: satellite associations. The D and G group chromosomes from cultured human lymphocytes exhibit single and multiple satellite associations when stained with silver. Unlike earlier methods this simple and highly repeatable procedure shows physical attachments between satellited regions of various acrocentric autosomes. After studying 1,000 satellite associations from 118 normal individuals, it was found that both single and multiple associations occur with frequencies that correlate with random expectancies.


[Animal experimental studies on bone regeneration following drilling of the bone]. Experiments in dogs showed that drilling at the surface of bone provokes damage which increases with the number of revolutions per minute. Polychrome sequence marking and histological tests demonstrated that filling up of bony defects started later with turbine-driven drills than with drills of 2,000 r.p.m. and has not even been completed after eight weeks. It is therefore recommended to abstain from using turbines in drilling at the surface of bone.


[Disc electrophoresis of urinary proteins. Changes after exertion]. The authors examined, by means of disc electrophoresis, urine, obtained from football players before and after a match of the championship of the "A" republican football group. Proteinuria after effort was manifested by a considerable excretion of albumin, but there were also serum proteins: postalbumin, transferin, gamma-globulins and fractions found at the place of alpha2-macroglobulins till a complete resemblence of serum electrophoresis. Electrophoresis of lactate-dehydrogenase revealed the presence only of two quick fractions, mainly LDH-1, which increased after effort as well. The fractions of nonspecific esterases were of predominantly tissue origin but after effort of serum origin. The results were discussed in connection with various mechanisms, which could cause proteinuria after physical effort.


Evoked potential decrements in auditory cortex. I. Discrete-trial and continual stimulation. In experiment 1 cats were exposed to sets of clicks (trials) with 1 min inter-trial-intervals to determine if the effects of repetitive stimulation on potentials evoked in the auditory cortex would be cumulative despite discrete-trial stimulation. Evoked potentials were averaged to give one average evoked potential (AEP) for each trial for each electrode; there were four cortical electrodes per subject. To test for dishabituation pawshocks were given between trials 60 and  61. Subjects were paralyzed to insure stimulus constancy. The latency and peak-to-peak amplitude of each component of each AEP was measured; significant amplitude decremented; and decrements were more frequent in components with latencies greater than 15 msec. A few amplitude increments and latency changes were also observed...


Evoked potential decrements in auditory cortex. II. Critical test for habituation. The goal of the present experiment was to rule out the hypothesis that evoked potential (EP) decrements during repetitive stimulation are due to a change in the subject's state; i.e., that the decrements are part of general, nonselective, non-specific decrement in all EPs as a result of a change in state during the course of the experiment. To this end, we obtained average evoked potentials (AEPs) to tone pips of two different frequencies; pips of one frequency were the repetitive stimuli, one and pips of the other frequency served as "test" stimuli. Before and again after a 15 min series of repetitive pips, AEPs to the repetitive stimuli and to the test stimuli were obtained. We found that from the beginning to the end of the 15 min repetitive-pip series, certain components of the AEPs to the repetitive stimuli decreased significantly in amplitude. However, comparable components in the AEPs to the test stimuli did not demonstrate significant decrements. These results suggested that the decrements were not part of a non-selective, non-specific, state-related decrement in all AEPs. However, the selective decrements could have been attributable to either of two aspects of the repetitive tone pips; their repetitive aspect, or their frequency. Because there were  two independent variables, a counterbalanced design was required. We found that regardless of which frequency was repetitive, there were significant decrements in the AEPs to the repetitive stimuli and smaller decrements (if any) in the AEPs to the test stimuli. This ruled out the frequencies of the tone pips as critical factors in the decrements; by the process of elimination the repetitive aspect of the stimuli remained as the critical variable. Only decrements that were specific to the repetitive stimulus were considered to be habituatory. All of the habituatory decrements were in components subsequent to the initial AEP components and had latencies in the range of 17--105 msec. In several cases, when subjects participated in a supplemental experiment, the same AEP components demonstrated habituatory decrements in both experiments.


Preliminary characterization of enzymes for reduction of naloxone and naltrexone in rabbit and chicken liver. The enzyme systems involved in the reduction of the narcotic antagonists, naloxone and naltrexone, were studied from the liver of chicken and rabbit. These secies were chosen because the relative amounts of diastereoisomeric products formed by enzymatic reduction between these species are so different; naloxone and naltrexone are converted in the chicken to 6alpha-naloxol and 6alpha-naltrexol, respectively, and in the rabbit to 6beta-naloxol and 6beta-naltrexol, respectively. In the present study, the enzymes for carrying out this reduction were found to require NADPH and to occur in the soluble fraction of liver homogenates. These enzymes were precipitated at 60-70% ammonium sulfate saturation for the chicken and at 50-60% for the rabbit. Differential sensitivity to inhibitors could be shown. For example, the rabbit enzyme was more sensitive to inhibition by morphine than was the chicken enzyme. The reverse order of sensitivity was seen with ketamine. These differences in properties between the chicken and rabbit liver enzymes indicate that the alpha- and beta-hydroxy product stereoselectivity arises from two different enzymes carrying out the reduction.


[EEG effects of sodium nitroprusside and hemodynamic references]. A study was performed on baboons (Papio papio) of various hemodynamic and EEG effects of sodium nitroprussiate (SNP), substance with powerful vasodilator action. Three different procedures of drug injection were used: (1) in isolation, either in doses close to those used in clinical practice or in high doses; (2) after neuroleptanalgesia (NLA); (3) after causing vasospasm. With isolated SNP, effects on blood pressure were unstable; in certain cases hypotension could be maintained only with toxic doses. Hypotension was facilitated when NLA had been previously induced. At low doses the cerebral blood flow (CBF) was practically unmodified, whereas at toxic doses it first increased then decreased. In animals under NLA, the CBF drop was only moderate, suggesting persistence of adequate spasm, CBF was significantly improved through SNP. The EEG did not undergo sizeable change at low SNP doses given in isolation; toxic doses always induced alterations with peculiar morphology, but developing only tardily after SNP perfusion was begun. This lag may indicate that hypotensive and toxic SNP effects are dissociated. These alterations suggest that screening of the EEG is necessary during prolonged administration of SNP, e.g., in surgical procedures. Combining NLA and SNP (at low doses) causes EEG deceleration, but no great alteration of the rhythms, as observed during other types of controlled experimental hypotension. Hence, cerebral circulation may be relatively well protected during SNP hypotension.


Visual evoked response in senile and presenile dementia. The visual cortical evoked response was studied in 19 patients suffering from a condition diagnosed as senile or presenile (type Alzheimer) dementia. When compared with a group of normal subjects used as reference group, the latencies of waves II through VI were increased or markedly increased in these patients, and the amplitudes of waves III and VI (and, less consistently, those of waves II and IV) were increased. This corroborates the sparse data on other types of dementia available in the literature.


Blink reflex in hemiplegia.

Blink reflex in hemiplegia. An electrophysiological study of the blink reflex was undertaken in 20 normal subjects and in 28 patients complaining of central facial palsy caused by unilateral hemispheral damage. In normal subjects, the latency, amplitude and organization of R1 and R2 responses are well known. Habituation of R2 responses occurred between 1 and 2 c/sec stimulation rate. R1 responses habituated at a higher stimulation rate (5 c/sec). In patients with unilateral hemispheral lesion, our results showed that changes in the blink reflex responses were bilateral. On the hemiplegic side the responses showed a decreased amplitude, while they were facilitated on the "normal" side. However, there was no change in latency of the two components of the reflex, on both sides. On the other hand, habituation of the late component occurred on the hemiplegic side for low stimulation rates: (0.5--1 c/sec), while on the "normal" side there was less habituation (3--4 c/sec), as compared with normal subjects. These results agree with those of experimental studies on cortical modulatory influences on brain-stem nuclei. They suggest a tactile origin of the two components of the blink reflex.


The grand mal pattern of Gibbs, Gibbs and Lennox. The "grand mal" pattern of Gibbs, Gibbs and Lennox was observed in records of 14 patients during the past 3 years. It occurs most commonly in those epileptic patients who suffer from primary generalized seizures of more than one seizure type and in whom akinetic attacks are the main clinical problem. In addition, there is usually some degree of intellectual limitations. The EEG features and clinical accompaniments of the discharge were described and since the term "grand mal" discharge is not appropriate, it is suggested that it be replaced by either "generalized repetitive fast discharge", as had been suggested in the European literature, or "beta band seizure pattern".


The EEG in centenarians.

The EEG in centenarians. 1. The EEGs of ten centenarians were described. 2. In most healthy centenarians, posterior dominant rhythms are within the lower part of the alpha range (average 8.62 c/sec). 3. Although slowing of the posterior dominant rhythm at age 100 is greater than in younger groups, there is no evidence in this study of a progressive decrease in frequency during the two decades between 80 and 100 years. 4. Most records of centenarians contained other abnormalities, either of diffuse slowing or of slow wave foci, the latter most often in the left temporal area. 5. The relationship of the observed EEG changes to the process of aging as distinct from reduced cerebral metabolism and blood flow resulting from disease is discussed.


Postnatal development of human brainstem potentials during the first year of life. Average brainstem potentials evoked by auditory stimuli were recorded from the scalp's surface of human newborns and infants, 6 weeks, 3 months, 6 months and 1 year of age as well as from normal adults. The auditory processes related to peripheral transmission (PT) and central transmission (CT) were shown to mature at differential rates during the first year of life. By the 6th week PT had reached the adult latency. In contrast, CT through the brainstem did not match that of the adult until approximately 1 year of age. In addition, the waveform of the brainstem evoked potential (BEP) displayed an independent time course for development. The adult configuration replaced the infantile response by 3--6 months. Despite considerable inter-subject variability in the BEP amplitude during the neonatal period, a general wave-form pattern could easily be delineated. Preliminary results revealed the BEP to be quite resistant to habituation following continuous stimulus presentation. Longitudinal follow-ups on several babies paralleled the transverse data.


A device used for study of postural reactions in the quadruped. A device used for study of postural reactions associated with placing movement in the quadruped is described. The apparatus consists of three basic components; a set of 4 strain gauge platforms on which the quadruped is trained to stand, a restraining device to keep the animal positioned over the strain gauge platforms and two mobile plates which mechanically stimulate the left or the right forelimb to produce the placing movement. The contact placing reaction consists of an early phase of preparatory positioning of the body (a postural adjustment) and a later phase of limb displacement, superimposed on a postural stance, during which the animal lifts its forepaw from the ground and then replaces it on top of one of the mobile plates.


Laboratory note. EEG changes after acute cerebral embolism. Embolism of the right middle cerebral artery regularly failed to induce clinical or electrical seizure activity during acute ischemia in primates. This negative correlation casts some doubt on the popular interpretation of seizures at the outset of clinical stroke as evidence of cerebral embolism.


Potassium-induced release of tritiated histamine from rat brain tissue slices. The release of exogenous histamine was studied by superfusing brain slices following incubation with the radiolabeled amine. Histamine was released in a calcium-dependent way by 40 mM potassium. This release was high in hypothalamus and striatum and low in hippocampus and cortex. Compound 48/80, a mast cell histamine releasing agent, also induced histamine release, but only from hypothalamic tissue slices. It is suggested that the potassium-induced release of accumulated exogenous histamine is mainly from glial cells.


Role of striatal dopamine in delayed neurotoxic effects of organophosphorus compounds. Mipafox administered to rats daily for 35 days produced ataxia and a reduction in the level of dopamine in the corpus striatum. Treatment with Leptophos for the same period produced slight motor dysfunction and a small but significant reduction in the level of striatal dopamine. Fenitrothion neither produced motor dysfunction nor changed the level of striatal dopamine. The cholinesterase activity of corpus striatum was inhibited by all the compounds. The results suggest the possible involvement of striatal dopamine in the delayed neurotoxic effects of certain organophosphorus compounds.


Peripheral blood T and B cell characteristics in a patient with severe combined immune deficiency (SCID) maintained in a gnotobiotic environment. Peripheral blood lymphocytes obtained at 24-30 months after birth from a male with X-linked severe combined immune deficiency maintained in a gnotobiotic environment were characterized by T and B cell surface markers. A high proportion (55-80 percent) of circulating lymphocytes bore surface IgM as detected by direct immunofluorescence. A receptor for the activated C3 complement component was detected on 27-47 percent of his lymphocytes. Only 4-12 percent of the peripheral blood lymphocytes formed spontaneous rosettes with sheep erythrocytes (E-R). In general, no blastogenesis was detected in lymphocyte cultures stimulated with pokeweed mitogen or phytohemagglutinin although transient slightly positive responses to both mitogens were occasionally observed. Incubation of lymphocytes with bovine thymosin Fraction V did not increase the percentage of E-R nor induce lymphocyte blastogenesis in the presence of phytohemagglutinin.


Projection of thalamic neurons to cat primary vestibular cortical fields studied by means of retrograde axonal transport of horseradish peroxidase. The two vestibular cortical projection areas in the anterior suprasylvian sulcus and post-cruciate dimple regions were defined by evoked potential technique in anaesthetized cats. The thalamic location of neurons with axon terminals in these fields was determined using the method of retrograde axonal transport of horseradish peroxidase. The ascending vestibular pathway appeared to be separated also at the thalamic level, where cells in the ventroposterolateral nucleus were found to project to the post cruciate dimple and cells in the posterior nuclear group to the anterior suprasylvian vestibular cortical fields.


Post-transfusion hepatitis: the role of hepatitis B antibody. Aliquots from units of blood previously transfused as part of a prospective post-transfusion hepatitis (PTH) study were rescreened for the presence of hepatitis B antibody (anti-HBS) to determine the effect of transfusion of such material. Anti-HBS was more commin in commercial blood. Infusion of anti-HBS was not associated with an increased or decreased risk of PTH, hepatitis B, or hepatitis B (HB) exposure. Receipt of anti-HBS did not modify the hepatitis which occurred. Receipt of large amounts of anti-HBS may be associated with an increased incidence of HB events. Preexisting anti-HBS was not only not protective against PTH, but more PTH (67% versus 40%) and hepatitis B (47% versus 12%) were seen in those patients with it.


[A course of pregnancy with herpes gestationis (author's transl)]. A 24-years old III.-para and III.-gravida was hospitalized in the 36th week of gravidity, with premature labour and distinct symptoms of herpes gestationis. It is reported in literature that the clinical picture of herpes gestationis is combined with a high rate of abortion and fetal anomalies. Therefore an intensive prenatal super vision was carried through with daily cardiotocographic controls and checkings of total estrogens in 24-hours-urine, also measurements of Alpha-Fetoproteins, human placenta lactogen and serumoxytocinase. After inducting labour because of maternal indication the patient gave birth to a healthy female infant (3720 g, 50 cm) with Apgar 10 in the 39th week of gravidity. The investigation of the placenta and all biochemical parameters did not point to an interference of gravidity. The present literature is discussed. Some authors doubt that herpes gestationis is met with a high rate of abortion, stillbirth and fetal anomalies as is assumed till now.


[Differential staining of hamadryas baboon chromosomes with Romanovsky-Giemsa dye (G-discs)]. The pattern of G-discs in the chromosomes of baboon (Papio hamadryas) was studied after staining by means of ASG method. On the basis of these data all the 20 pairs of autosomes and sex chromosomes were identified. According to the distinctness of the discs all the chromosomes were classified into 3 groups: well differentiated, faintly differentiated and moderately differentiated. The most distinct pattern of discs was obtained in slightly spiralized chromosomes. Dimorphism of the disc pattern in homologous chromosomes was observed, which is, possibly, indicative of their different functional activity.


[Chromosome identification in Macaca arctoides based on study of the differential staining pattern (G-discs). Comparison with Papio hamadryas G-discs]. Linear chromosome differentiation pattern of Macaca arctoides was studied using the Romanovsky-Gimsa stain (G-banding). The possibility of the identification of all chromosome pairs according to these data is demonstrated. The phenomenon of the irregular intercellular and chromosome differentiation is found out and a suggestion is made about its relation to a real mosaic functional activity of cell genomes. A great similarity in linear chromosome differentiation for 2 from 42 studied chromosome types of Macaca arctoides (genus Macaca) and Papio hamadryas (genus Papio) is shown. On the basis of these data it is supposed that in the divergency process of these species the chromosome microaberrations and molecular mutations played an important role, while large chromosome reconstructions were of no importance. The similar G-banding pattern of both metacentric chromosome arms in two species is found and this suggests an isochromosome mechanism of their occurance.


[Diagnostic and therapeutic problems in retarded intrauterine development]. The present paper reports on the deliveries of 16 infants exhibiting chronic intrauterine growth retardation, reflected by a birth weight between 900 and 2000 gm. All babies were delivered by caesarean section. The validity of prenatal supervising methods is demonstrated with reference to termination of pregnancy.


[Various banding simultaneously obtained on the same slides, after treatment by BrdU (author's transl)]. A treatment by BrdU during 24 hrs, followed by a treatment by thymidine, during 30 hrs, induces in the same preparations, many types of chromosome banding R-, Q-, H-bands, and several types of asymmetry. This indicates that the BrdU is not incorporated only in some parts of the karyotype, but, on the contrary, any where, and then induces different banding according to the site of its incorporation. Above all, the type of band is determined by the period of the phase S in which the cell was at the time of the treatment by BrdU. Thus the coexistence of many types of banding may be explained here by the great variability of the cellular cycle of the lymphocytes in culture.


Dissociation of anticomplementary and adjuvant properties of proteins derived from cobra venom. The ability of preparations of cobra factor (CoF) to convert C3 via the properdin pathway (anticomplementary activity) has been demonstrated to be unrelated to its ascribed ability to convert a tolerogenic signal into an immunogenic stimulus (adjuvant activity). It has been shown that CoF preparations which have become inactive with regard to anticomplementary activity still retain full adjuvant properties. Further, when both activities are present in preparations of CoF, they can be separated by gel filtration chromatography into a fraction with approximate molecular weight 150,000 which contains anticomplementary activity and a fraction with approximate molecular weight 20,000, which contains adjuvant properties.


The materno-foetal transfer of carrier protein sensitivity in the mouse. The anti-dinitrophenol (DNP) antibody response was examined in the 18-week-old offspring of female mice primed to bovine serum albumin (BSA) and in control unprimed animals. Comparisons were made of the response to DNP-BSA, DNP-chicken gamma-globulin (CGG) and DNP-purified protein derivative (PPD) after all offspring had been primed with DNP-CGG at 12 weeks of age. An enhanced anti-DNP antibody response was found to DNP-BSA in mice whose mothers had received BSA. It is suggested that this phenomenon demonstrates the transfer of carrier protein sensitivity from mother to foetus.


Urinary flow rate in benign prostatic hypertrophy. Multiple urinary flow measurements were recorded on 12 men without bladder outlet obstruction, ten men with a clinically doubtful diagnosis of bladder outlet obstruction due to benign prostatic hypertrophy, 29 men with a proven clinical diagnosis of bladder outlet obstruction due to benign prostatic hypertrophy and eight men before and after prostatic surgery. All were over 50 years of age. Mean peak flow rates for non-obstructed men varied from 11.8 to 35.0 ml per second and the minimum threshold for mean peak flow rate in these subjects was 15.0 ml per second for voided volumes of 200 ml and above. The vast majority of subjects with obstruction had mean peak flow rates below 15 ml per second. Mean peak flow rates reverted to normal after prostatic surgery. Our findings justify the use of multiple determinations of peak flow rate as a routine screening examination in subjects who may have bladder outlet obstruction due to benign prostatic hypertrophy.


Antibodies to radiographic contrast agents. Induction and characterization of rabbit antibody. An allergic, immunologic mechanism for certain adverse reactions to radiocontrast agents has been postulated on the basis of clinical and theoretical considerations. One obstacle to the acceptance of this allergic theory has been the inability to demonstrate significant antibodies with specificity for these agents. We attempted the induction of antibodies in the rabbit using analogues of radiocontrast media. The synthesis of four different analogues of commonly used radiocontrast agents allowed for testing the haptenic potential of various portions of the contrast medium molecule. Each analogue was conjugated to a carrier protein, emulsified in a suitable adjuvant, and repeatedly injected into several rabbits. Optimal immunization conditions were selected to increase the probability of antibody induction. Specific antibody was produced in good titer to each of the four haptens and in all injected rabbits. Antibodies were characterized by means of precipitation assays and radioimmunoassay using contrast agents labeled with 125I. Antibody concentrations ranged from 0.23 to 2.7 mg/ml and varied with interval and frequency of booster injections. In addition to antibodies specific for the contrast media analogues, antibodies to the carrier protein were also detected. Antibodies were of the IgG class and in selected sera were 83-92% precipitable. The induction of antibodies to radiocontrast agents supports the allergic theory of contrast medium toxicity.


Comparative field cage tests of the population suppressing efficiency of three genetic control systems for Aedes Aegypti. Cycling populations of Aedes aegypti were set up in cages and managed in such a way that the populations had a maximum of threefold recovery potential in response to control measures. Into three such populations daily releases were made of males which had been chemosterilised, or were double translocation heterozygotes (T1T3) or T1T3 with sex ration distortion (DT1T3). Eradication of the populations was achieved with all cases, but the rate of suppression was markedly slower with T1T3 than the other two systems, with which the rates were similar. T1T3 and DT1T3 releases introduced considerable inherited genetic loads into the target populations. The results were in general agreement with computer predictions.


Bacteriophage Mu-1-induced mutation to mutT in Escherichia coli. Of approximately 10,000 independent phage Mu-1 lysogens, 3 had a mutator phenotype. One (mutation designated mut-49) resembled mutT1 in the frequency and types of mutations induced. mut-49 was mapped between leu and ace and was not separable from the Mu prophage. mut-49 was recessive and did not complement mutT1. mut-49, like mutT1, did not increase the reversion of the frameshift mutation lac Z (ICR48). mut-49 and mutT1 induced the same two classes of trpA78 revertants, indicating that mut-49 induced adenine-thymine leads to cytosine-guanine transversions. The results support previous work indicating that the mutational specificity of mutT is gene and not allele specific.


Immunofluorescent polar tips of Rhizobium japonicum: possible site of attachment or lectin binding. Rhizobium japonicum USDA 31 demonstrated marked polarity by binding homologous fluorescent antibody (FA) heavily on one end of the cell. FA prepared against R. japonicum strains 110 and 138, and against R. trifolii TA1 cross-reacted with strain 31 only in the polar tip region. No polar immunofluorescing tips could be seen with FA against two other strains of R. japonicum or with those against several unrelated microorganisms. Common antigens localized only in a polar region were seen in many rhizobia stained with R. japonicum 31 FA: 22 of 23 strains of R. japonicum, 10 of 17 strains of R. trifolii, 3 of 7 strains of R. melitolii, 3 of 6 strains of R. phaseoli, and 3 of 9 strains of R. leguminosarum had some cells with detectable polar tips. The proportion of R. japonicum 31 cells with polar tips was high throughout the growth cycle. Polar tip staining was not affected by drastic cell treatments. A function was proposed for the polar tip region as a site for attachment. R. japonicum 31 cells attached to each other in a tip-to-tip fashion and endwise to fungal hyphae with the polar tip in contact with the hyphal wall. Binding of fluorescein isothiocyanate-labeled soybean lectin to certain strains of R. japonicum gave additional evidence of polarity. Polar binding of both antibody and lectin may provide insights into relationships between rhizobia and roots of host legumes.


Action of fibrin-stabilizing factor on cold-insoluble globulin and alpha2-macroglobulin in clotting plasma. Experiments were performed to investigate whether proteins other than fibrin are substrates for activated fibrin-stabilizing factor (FSF, blood coagulation Factor XIII, plasma transglutaminase) in clotting whole plasma. Three fluorescently labeled polypeptides were identified in serum prepared by clotting normal, but not FSF-deficient, plasma in the presence of the fluorescent amine, N-(5-aminopentyl)-5-dimethyl-aminonaphthalene-1-sulfonamide (dansylcadaverine). The major labeled polypeptide had a Mr (estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate) of 1.6 times 10(5) and was found in the protein fraction precipitated by 33 to 50% saturated ammonium sulfate. The second had a Mr of 2.0 times 10(5), was found in the protein fraction insoluble in 33% saturated ammonium sulfate, and was precipitated by gamma-globulin directed against cold-insoluble globulin. The third had a Mr of 1.1 times 10(5) and was precipitated by 33 to 50% saturated ammonium sulfate. All three polypeptides were found in the first protein peak when labeled serum was chromatographed on Sephadex G-200. The immunoprecipitin arc containing alpha2-macroglobulin was fluorescent when labeled serum was analyzed by immunoelectrophoresis. These results indicate that alpha2-macroglubulin, cold-insoluble globulin, and an unidentified third protein with a subunit of Mr = 1.1 times 10(5) are transamidated by FSF in clotting plasma. The concentration of cold-insoluble globulin was decreased in serum formed at 37 degrees from normal, but not from FSF-deficient, plasma. The depletion of cold-insoluble globulin in normal serum was partially blocked by clotting in the presence of dansylcadaverine and completely blocked by clotting in the absence of calcium ions. Sera formed at 2 degrees from both normal and FSF-deficient plasma contained less cold-insoluble globulin than plasma. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of clots formed at 2 degrees demonstrated cross-linking of cold-insoluble globulin to fibrin in the normal, but not the FSF-deficient, sample. The serum concentration of alpha2-macroglobulin was the same as the plasma concentration irrespective of the conditions of clotting. Thus, the experiments suggest that FSF catalyzes the cross-linking of cold-insoluble globulin (but not alpha2-macroglobulin) to fibrin in clotting plasma.


The resurfacing of adult rabbit articular cartilage by multiple perforations through the subchondral bone. The repair of articular cartilage removed from the femora of adult rabbits at the knee was studied. When multiple perforations were made through the subchondral bone a cartilaginous material, staining heavily with safranin O, first filled the holes. This material resembled hyaline cartilage, and by twelve months there was complete resurfacing of the joint, although the material lost its hyaline appearance after eight months and at one year it resembled dense collagenous tissue.


Flow microfluorometric analysis of sperm DNA content: effect of cell shape on the fluorescence distribution. The DNA content of individual sperm from populations of acriflavine-stained cells was investigated by analysis of fluorescence frequency distributions obtained with high-resolution flow-systems instruments. Sperm with spherical or cylindrical heads from three mollusk species produce narrow, symmetric fluorescence distributions. Flat sperm heads from six eutherian species produce asymmetric distributions consisting of a peak with a lateral extension to higher fluorescence values. The unexpected shape of these distributions was shown to be due to the flat geometry and high refractive index of the sperm heads in conjunction with the orthogonal axes of flow, excitation, and detection in the flow-systems instruments. The theoretical and experimeytal results indicate that the lateral extension can be eliminated either by controlling the sperm orientation with planar flow conditions or by accounting for sperm orientation by means of orientation sensing.


[Chronic knee instability. Technics and results of ligament plasty in sports injuries]. Chronic laxity of the knee in sportsmen is limited in practice, owing to the mechanisms of blockage of rotation, to lesions of the medial and anterior cruciate ligaments, associated or not with tears of the meniscus. Full assessment is essential to draw up a full therapeutic plan. It is very difficult, even when clinical examination is completed by excellent XRays. The great technical facility of operations of musculo-aponeurotic transposition, has led surgeons to discard plastic operations on the ligaments. They do, however, give the best chance of return to sporting activity. Two techniques are discussed, one a plastic of the medial ligament, the other designed to palliate rupture of the anterior cruciate ligament. These operations are strong and precise straightaway but the prognosis depends mainly on the number of structures affected.


Specific immunological test for the rapid identification of members of the genus Histoplasma. A sensitive and specific immunological method was developed for rapid identification of the mycelial forms of Histoplasma capsulatum var. capsulatum, H. capsulatum var. duboisii, and H. farciminosum and for separation of these pathogenic fungi from morphologically similar hyphomycetes and other fungal pathogens. This method is based on the fact that all of the Histoplasma spp. produce H and M histoplasmin antigens, whereas the other fungi do not. Inocula consisting of heavy mycelial growth from a pure, full-grown culture were transferred into flasks containing small volumes of brain heart infusion broth. These cultures were placed on a shaker and grown at 25 C. Using the micro-immunodiffusion technique and antisera containing antibodies to H and M precipitinogens, we detected exoantigens in 3-day-old brain heart infusion culture supernatants concentrated 25 and 50 times. The ability of the procedure to identify Histoplasma spp. was evaluated by testing 96 unknown mycelial cultures that grossly or microscopically resembled Histoplasma spp. Three- and six-day-old concentrated culture supernatants prepared from each unknown were tested against rabbit anti-Arthroderma tuberculatum, Chrysosporium keratinophilum, H. capsulatum var. duboisii, and Corynascus (Thielavia) sepedonium sera and human histoplasmosis case serum. Each unknown was also identified by conventional laboratory procedures involving cultural and, where necessary, in vivo studies. In the comparative evaluation the immunological test was observed to be 100% sensitive. It permitted the accurate generic identification of the Histoplasma spp. within 5 days, in contrast to the average of 33 days required by the routine mycological procedure.


Glomerulonephritis mediated by antibody to glomerular basement membrane. Immunological, clinical, and histopathological characteristics. A prospective study was undertaken to establish the incidence of glomerular basement membrane (GBM) antibody-mediated glomerulonephritis and its histopathological characteristics in a clinical group of patients presenting with renal disease. Biopsies from 43 of 409 consecutive patients technically satisfactory for direct immunofluorescent (IF) examination had diffuse and generalized linear localization of host immunoglobulin (Ig); two other badly scarred kidneys tested negative to IF although GBM antibodies were eluted. Confirmatory evidence of GBM antibody-mediated disease in these patients came from whole kidney or biopsy elutions (15 patients), serologic assays for circulating GBM antibodies by indirect IF (9 of 38 patients), radioimmunoassay (26 of 34), and hemagglutination (31 of 32). Although sera were not tested from six patients, circulating antibodies were demonstrated by some test in 36 of 39 of the remainder. Histologically, half of the patients had minor and nonspecific glomerular abnormalities or mild focal proliferative glomerulonephritis. More severely involved kidneys had focal necrotizing (17%), rapidly progressive (7%), and chronic, usually sclerosing, glomerulonephritis (27%). Clinical courses of these patients comparably were quite variable, ranging from indolent microhematuria and/or gross hematuric bouts to progressive renal failure; nephrotic syndrome was observed in 11 patients. GBM antibody-mediated glomerulonephritis may be a relatively mild disease with apparently stable renal function, although 16 patients have experienced functional deterioration, and 11 have progressed to dialysis, renal transplantation, or death.


Quantitative analysis of pregnancy-associated plasma proteins in human placenta. By immunochemical methods and simultaneous measurements of several normal plasma proteins, human placenta was shown to contain elevated quantities of four pregnancy-associated plasma proteins (PAPP's). In the order of increasing amounts, PAPP-A, PAPP-C, PAPP-B, and human chorionic somatomammotropin (PAPP-D) all were present in placenta extracts in quantities greater than could be expected on the basis of their content in maternal blood. In sharp contrast, the placental content of pregnancy zone protein could be entirely accounted for by the maternal plasma present in the placenta. All of the PAPP's appeared to be readily extractible from placental tissue with buffered saline, the large bulk of them being solubilized in the first extraction procedure. However, absorption studies indicated that appreciable quantities of the PAPP's were still present in the insoluble placental residue after 12 sequential extractions with saline. The chorioamniotic membranes were not significantly enriched in any of the PAPP's. Immunochemical analysis of unwashed placental tissue extracts for the PAPP's IgA, and IgM (maternal blood derived), as well as albumin and transferrin (maternal and fetal blood derived), permitted calculations to be made of the amount of blood and PAPP's in placenta. On the basis of these data, it was roughly estimated that a 400-g placenta (wet weight) would occupy 312 ml in volume, and would contain 144 ml of blood. Of this blood, 36 ml would be derived from the mother.


Metabolism of thyroxine-binding globulin in man. Abnormal rate of synthesis in inherited thyroxine-binding globulin deficiency and excess. It has been previously suggested that inherited thyroxine-binding globulin (TBG) abnormalities in man may be due to mutations at a single X-chromosome-linked locus controlling TBG synthesis. However, abnormalities in TBG degradation have not been excluded. The availability of purified human TBG and its successful labeling with radioiodide allowed us to examine such possibility. Human TBG was purified by affinity chromatography, labeled under sterile conditions with 131I or 125I,, and mixed with [125I]thyroxine (T4) or [131I]T4, respectively, before their intravenous injection. Blood and urine samples were collected over a 10-day period, and the turnover parameters were calculated. In eight normal volunteers mean values +/-SD for TBG and T4 respectively, were as follows: Half time (t1/2) 5.3 +/- 0.4 and 7.0 +/- 0.6 days; distribution space (DS) 7.2 +/- 1.0 and 10.8 +/- 1.2 liters; and total daily degradation (D) 0.211 +/- 0.053 and 0.088 +/- 0.011 mumol/day. In all subjects, t1/2 of TBG was shorter than that of T4; and the DS was smaller. 2.4 mol of TBG was degraded for each mole of T4. In five of six subjects from four families, comprising hemizygous and heterozygous carriers of TBG absence, decrease, and excess, the t1/2 and DS for TBG were within the normal range. The D of TBG was proportional to the serum concentration of the protein. Changes in the T4 kinetics in these patients were compatible with euthyroidism and with the known alterations in the extrathyroidal T4 pool associated with the changes in serum TBG concentration. A striking decrease in the t1/2 of TBG was found only in a patient with acquired diminution in TBG concentration and in patients with thyrotoxicosis or other conditions apparently unrelated to thyroid dysfunction. TBG t1/2 was 2.5 days in a patient with multiple myeloma and 3.6 days in two patients with thyrotoxicosis. Decreased TBG t1/2 was also observed in three of six patients with nonthyroidal pathology and was associated with an increase in TBG D disproportionate to their level of serum TBG. These studies indicate that changes in TBG concentration in patients with X-chromosome-linked TBG abnormalities are due to alterations in its rate of synthesis. In other conditions, abnormalities of TBG degradation and/or rate of synthesis may be found.


Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus. Homogenized tissue from the frontal cortex of normal human brains obtained at postmortem examination was used to absorb lymphocytotoxic antibody from the serum of six patients with systemic lupus erythematosus (SLE). Four absorptions of all of the SLE sera with equal volumes of homogenized brain tissue at 4 degrees C depleted their cytotoxic capacity more than 90%. Three of the six sera, however, retained some lymphocytotoxicity despite extensive brain absorption. Absorbed lymphocytotoxic antibodies were eluted from brain tissue absorbents at 37 degrees C. Cytotoxicity of the brain eluates was blocked by antibodies to human IgM (mu-chain specific) but not anti-IgG. The unabsorbed SLE sera, brain-absorbed sera, and brain eluates were equally cytotoxic to T (thymus-derived) and B (bone marrow-derived) cells fractionated from normal human peripheral blood lymphocytes. Thus, the lymphocytotoxic antibodies in SLE serum exhibit no preference for circulating human T cells. An analysis of the clinical records of 40 patients with SLE whose serum cytotoxic capacity had been determined revealed that circulating lymphocytotoxicity is greater in sera of patients with central nervous system (CNS) manifestations than in other SLE patients. This observation suggests a possible role for brain-reactive lymphocytotoxic antibodies in the development of CNS disease in SLE.


Markovian analysis of cervical cell images. Markovian analysis is a method to measure optical texture based on gray-level transition probabilities in digitized images. Experiments are described that investigate that classification performance of parameters generated by Markovian analysis. Results using Markov texture parameters show that the selection of a Markov step size strongly affects classification error rates and the number of parameters required to achieve the maximum correct classification rates. Markov texture parameters are shown to achieve high rates of correct classification in discriminating images of normal from abnormal cervical cell nuclei.


Automatic leukocyte classification using cytochemically stained smears. A leukocyte classification algorithm suitable for automated differential counting has been developed for blood smears stained with a new three-component cytochemical stain which has relatively narrow absorption bands centered at 460, 540 and 640 nm, respectively. The classification procedure is the result of a pattern recognition experiment using a sample of 223 leukocytes distributed evenly over the five normal cell types. The basic data for each cell were three digital microscopic images obtained with narrow band illumination at the above central wavelengths using a TV-digitizer system interfaced to a PDP-15 computer. The classification algorithm involves a sequential decision procedure utilizing five pattern features computed from the intensity histograms of the green and blue digital images. Thus the number of arithmetic operations and the number of computer memory words necessary to perform the classification into one of the five normal white blood cell types are both proportional to n where n is the number of gray levels into which the intensity scale is divided. In this experiment, n equals 256. Comparison of our results with work of others on smears prepared with Romanowski-type stains indicates that such narrow-band, spectrally well separated cytochemical multiple stains can permit the use of algorithms which are approximately ten times faster.


Quantitative fluorescence spectrophotometry of acridine orange-stained unfixed cells. Potential for automated detection of human uterine cancer. After staining with acridine orange (AO), the nuclei of unfixed cells from the human female genital tract exhibited the same fluorescence behavior previously observed for human and murine leukocytes and mouse ascites tumor cells. With staining conditions chosen to assure saturation of the green-fluorescing AO-nucleic acid complex in normal cells, corrected fluorescence emission spectra were recorded from the entire nucleus of 341 cells taken from 32 normal and 28 abnormal patients. Intensity of the recorded spectra was expressed in phosphor particle units, a fixed arbitrary unit of fluorescence intensity, to display intensity differences among the spectra from the various cell types. In all abnormal samples, one or more cells were found with 530-nm nuclear fluorescence intensity considerably greater than the maximum intensity recorded from normal cells. Determination of the adequacy of 530-nm nuclear fluorescence intensity as a criterion for cancer detection requires additional investigation. Additional criteria, if needed, may be supplied by the metachromasy of AO-stained unfixed cells.


G-band patterns of swine chromosomes.

G-band patterns of swine chromosomes. A simple inexpensive and reproducible G-banding technique for swine chromosomes is described that permits precise identification of individual chromosome pairs. The G-bands were obtained by either SSC (saline-citrate-solution), or trypsin pretreatment methods.


Studies on human monocytes with a multiparameter cell sorter. Suspensions of human lymphocytes and monocytes separated by the Ficoll-hypaque method from the peripheral blood show a Coulter volume distribution, measured with a multiparameter cell sorter, characterized by a minor peak at 500 mu3, containing 5-15% of the cells, and a major peak at 200 mu3. Using fluorescent latex particles we have found that the monocytes, the cells that ingest the latex particles, all lie in the 500 mu3 peak; conversely, all of the cells in the 500 mu3 peak are monocytes. When the cell suspensions are incubated, the monocytes increase both the average volume and in absolute numbers. The number of monocytes approximately doubles during 3 days of incubation, when it reaches its maximum value. At that time we have found that all of the monocytes lack receptors for sheep red blood cells and all possess receptors for human gamma-globulin. The increase in monocyte number appears, therefore, to arise from the enlargement of "monocyte presursors" that resemble lymphocytes in volume and resemble both the monocytes and the B lyphocytes with respect to surface sheep red blood cell and human gamma-globulin receptors.


Combined blood cell counting and classification with fluorochrome stains and flow instrumentation. A multiparameter flow cytophotometer was used to count and classify fixed human blood cells fluorochromed with a mixture of ethidium bromide (EB), brilliant sulfaflavine and a blue fluorescent stilbene disulfonic acid derivative (LN). The system measures light scattered by the cells and absorption at 420 nm for all cells. In addition, nuclear EB fluorescence (540 leads to 610 nm) and cytoplasmic fluorescence from LN (366 leads to 470 nm), brilliant sulfaflavine (420 leads to 520 nm) and EB exicted by energy transfer from LN (366 leads to 610 nm) are measured for all nucleated cells. This information is sufficient to perform red and white blood cell counts and to classify leukocytes as lymphocytes, monocytes, basophils, eosinophils or neutrophils. Light scattering and/or nuclear and cytoplasmic fluorescence values may be further analyzed to obtain the ratio of immature to mature neutrophils. Counts produced by the system are in reasonable agreement with those obtained by electronic cells counting and examination of Wright's-stained blood smears; some discrepancies appear to be due to systematic errors in the manual counting method.


Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells. Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.


A new type of two-color fluorescence staining for cytology specimens. A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a  blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.


Dimethyl sulfoxide-lead tetraacetate method for histochemical oxidation of polysaccharides. Oxidation of fixed tissues and unfixed peripheral blood smears by 1% (w/v) lead tetraacetate in dimethyl sulfoxide for 10 to 60 min resulted in facile induction of tissue carbonyls readily demonstrable with Schiff's reagent and by sodium bisulfite addition reaction, followed by toluidine blue staining at controlled pH. Tissue carbonyls represented a broad range of oxidation-labile substrates and included neutral polysaccharides (glycogen). The oxidation procedures were not destructive to tissues and were characterized by technical simplicity and consistent reproducibility, thus affording a substantial improvement over the hitherto used methods of histochemical oxidation by lead tetraacetate.


Ultrastructural histochemical localization of acid phosphatase in salivary glands of Drosophila melanogaster. The ultrastructural histochemical localization of acid phosphatase in salivary glands of third instar larvae of Drosophila melanogaster has been studied. Using Gomori's lead phosphate method for acid phosphatase detection, the optimal incubation time in the reaction medium was determined to be 30 min. When glands having wild-type acid phosphatase activity are incubated for this time, deposition of the final reaction product is observed in essentially every lysosome and artifactual staining is minimal.


Resistance to tumor growth mediated by Listeria monocytogenes. Destruction of experimental malignant melanoma by LM-activated peritoneal and lymphoid cells. A murine experimental model of nonspecific tumor destruction mediated by cells activated by Listeria monocytogenes (LM) is described. B16 melanoma growth is prevented or suppressed in the syngeneic host when tumor cells are inoculated in contact with viable LM. In vitro, cultured B16 cells are destroyed by LM immune peritoneal or splenic cells in the presence of the bacterial antigen(s). Activation of LM immune cells in vitro is immunologically specific. Replacement of LM by sheep red blood cells or bovine serum albumin in the in vitro cultures aborts the cytotoxic effect. Further, no tumor cell killing is obtained when thioglycollate-induced or normal peritoneal cells are substituted for LM immune cells in the in vitro cultures. Normal spleen cells in the presence of LM are weakly cytotoxic for B16 cells. Normal peritoneal cells plus LM or LM alone are not. Elimination of thymus derived "T" cells by anti-theta C3H or rabbit anti-mouse brain serum (RAMB) abrogated the cytotoxic effect. Therefore, LM-induced tumor destruction probably occurs through nonspecific mechanism(s) consequent to activation of host "T" cells by specific immune reactivity to LM antigen(s).


Modulation of help and suppression in a hapten-carrier system. Provision of beta-galactosidase (GZ) under defined conditions of dose and time can either help or suppress a subsequent response to trinitrophenyl (TNP)-GZ in CBA/J mice. The optimal helper effect occurs when 10(7) spleen cells from mice primed 9 or more days previously with 10 mug GZ are adoptively transferred to irradiated recipients which are than challenged with 10 mug TNP50GZ. Optimum suppression results from the transfer of spleen cells from mice primed 3 days previously with 100 mug GZ and challenge of recipients with TMP150GZ. Both help and suppression are carrier-specific and mediated by T cells. In experiments where helper or suppressor cells were mixed with normal cells, the anti-TNP response was proportional to the number of primed cells transferred. The results point to a wave of suppression as the initial event after immunization, which is succeeded by period in which the helper effect dominates.


Complement-induced histamine release from human basophils. II. Mechanism of the histamine release reaction. The activation of human serum complement by incubation with zymosan generates C5a which releases histamine from autologous basophils. The characteristics of the C5a-induced histamine release were investigated. It is similar to IgE-mediated reactions in requiring Ca++ and in being inhibited by EDTA. However, it has marked differences from IgE-mediated reactions. C5a, at all concentrations, released histamine completely in less than 2 min. The C5a reaction has a narrow pH optimum that antigen-induced release and occurs well at 17 degrees to 37 degreesC but not at 0 degreesC. The optimal reaction temperature is 25 degrees to 30 degrees C. Unlike the antigen-induced release, no two-stage activation with C5a for the release of histamine could be demonstrated. There was additive release between C5a- IgE-mediated reactions. Leukocytes could be desensitized to the C5a-mediated reaction by 1) incubating the cells at 37 degrees C for 45 min, 2) pretreating the leukocytes with activated serum in the presence of EDTA, and 3) adding the activated serum to the leukocytes at 0 degrees C before transferring to the optimal reaction temperatures. Cells desensitized to the complement-induced release have normal reactions to IgE-mediated histamine release. In parallel experiments, cells from allergic donors desensitized for IgE-mediated reactions by incubation with antigen under sub-optimal conditions release histamine normally upon the addition of C5a. The results indicate that histamine release by C5a involves a mechanism of basophil activation that is different from the pathway involved in the IgE-induced reaction.


Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response. Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.


A new alloantigenic system associated with the Mls locus in the mouse. An antiserum prepared by injecting C3H/HeJ mice with CBA/J tissue has been shown to react with cell surface components that are not part of any previously described system of serologically detectable alloantigens. The antiserum, which is designated AST-101, acts selectively in cytotoxic tests carried out with lymphoid cells, killing B cells, but not T cells. Phagocytic cells found in peritoneal exudates are also killed by AST-101 and complement in vitro; the sensitivity of other cell types has not been determined. Strain distribution does not indicate any association of the AST-101 system with H-2, Ly, or Thy systems; genetic analysis reveals close linkage with the mouse minor MLC-stimulating (Mls) locus. Serologic analysis also points to a close association between antigens reactive with AST-101 and the products of the Mls genes.


Response to antigenic determinants of Neisseria meningitidis lipopolysaccharide investigated with a new radioactive antigen-binding assay. Adaptations of the Farr technique have resulted in a specific and reproducible radioactive antigen-binding assay for antibodies directed against the lipopolysaccharide (LPS) of Neisseria meningitidis. LPS was intrinsically labeled with 14C acetate during 16-hr growth in a modified Frantz media, extracted by hot phenol-H2O, and purified by dialysis, ultracentrifugation, and ethanol precipitation. LPS, which aggregates in aqueous solutions, was maintained in a monomeric form in 3% sodium deoxycholate (NaD) as determined by gel filtration on Sephadex G-75. Since NaD is insoluble in (NH4)2SO4, polyethylene glycol, 20%, was used to precipitate immunoglobulins of all three major classes.


SPECIFIC positive and negative selection of rat lymphocytes reactive to strong histocompatibility antigens: activation with alloantigens in vitro and in vivo. This study compares the functional properties of rat thoracic duct lymphocytes (TDL) after stimulation with strong alloantigens of the major histocompatibility complex (MHC) either in vitro in preparative mixed lymphocyte interactions (MLI) or in vivo in systemic graft-vs-host (GVH) reactions. Comparisons were made of PHA responses and reactivity to the specific priming haplotypes or to third party haplotypes in analytical MLI and in GVH reactions either before or after the activated populations were "parked" in syngenetic T cell-deprived (B) rats. These comparisons can be summarized as follows: 1) TDL populations primed in bulk MLI cultures (MLI-TDL) slowed some evidence of specific positive selection when tested immediately; MLI responses to specific alloantigens were both relatively large and accelerated in tempo, whereas responses to third party alloantigens were diminished but also accelerated in tempo. Specific GVH responses were more marked than in third party recipients but they were also decreased relative to normal, and displayed an abberant dose/response slope. MLI-TDL populations tested after they had been stored in syngeneic B rats showed clear evidence of stable-specific positive selection; specific MLI and GVH responses were enriched relative to third party responses and also in comparison to normal, unselected TDL populations. This finding indicates that GVH and MLI reactivity are probably both functional capacities of the same lymphocyte subpopulation since positive selection by one function (MLI) also enriched for a second (GVH). 2) Parental strain TDL activated in vivo in the systemic GVH reaction in irradiated F1 animals and recovered from the thoracic duct 3 to 4 days later (late GVH-TDL) consisted mainly of blast cells, however, in contrast to MLI-TDL these populations showed no evidence of positive selection when tested before or after parking in B rats. MLI responses to specific alloantigens were minimal, and greatly reduced in magnitude compared to normal. GVH responses to specific haplotypes could be detected, but these were not enriched compared to normal, despite the content in the late GVH-TDL populations of a significant proportion of blast cells presumably activated by host alloantigens. 3) Early collections (less than 40 hr) of parental strain GVH-TDL collected from F1 recipients contained no blast cells and showed impressive degrees of negative selection; they were markedly depleted of both GVH and MLI activity to specific alloantigens but displayed normal reactivity to third party alloantigens. Moreover, specific negative selection was persistent in these populations parked for several weeks in B rats, and indication that a specific subpopulation of reactive cells had been physically eliminated. 4) PHA responses of both MLI- and GVH-activated TDL populations tested either before or after parking in B rats were approximately normal on a per T cell basis...


Effect of proteolytic digestion on the structure and function of human properdin. Human properdin (P) was found to be sensitive to the action of trypsin, chymotrypsin, pepsin, and Streptomycetes caesipitosus protease. Incubation of P with these enzymes resulted in loss of its functional activity and the production of antigenically deficient components compared to untreated P. Upon incubation with trypin, P was initially cleaved into a minor fragment and a major fragment. Further degradation ot the fragments occurred with prolongation of inculation time. The minor fragment was highly susceptible to further proteolysis compared to the major fragment which contained the carbohydrate moiety of the molecule. SDS-polyacrylamide gel electrophoretic analysis of trypsin-digested P suggested that the subunit polypeptide chains were initially cleaved at similar points to produce the major and minor fragments. The sedimentation velocity of the major fragment was higher than that of the intact molecule. The implications of these observations of the configuration of P are discussed.


Characterization of T and B antigen-binding cells for beta-galactosidase. I. beta-galactosidase-binding cells in the thymus and spleen of normal mice. The results reported in this paper demonstrate that the enumeration of cells binding beta-galactosidase (Z) as an antigen, revealed by subsequent substrate hydrolysis, is an excellent method for the detection and study of antigen-binding cells (ZBC). The binding found is specific and is restricted to a small number of lymphocytes that bind a large number of Z molecules via surface receptors. Such ZBC were found at mean frequencies of 150 per 10(6) in the thymus and 200 to 300 per 10(6) in the spleen. The binding cells of both organs were heterogenous with individual ZBC binding from 10(5) to 10(6) molecules of enzyme as determined by substrate hydrolysis, although this might well be an overestimate of the number of actual receptors. The profiles for the frequency of ZBC binding different numbers of molecules were nearly identical for thymus and spleen, in contrast to descriptions of the binding of many other antigens. Receptors responsible for Z binding appear to be superficially located on the cell since they are trypsin-sensitive to a large extent and are not increased by fixation.


Inhibition of stimulation in murine mixed lymphocyte cultures with an alloantiserum directed against a shared Ia determinant. Pools of high titered alloantisera were raised by immunizing (B10.A/SgSn X A/WySn)F1 mice with C57BL/10Sn(B10) spleen cells. This serum (F1 anti-B10), when added to one-way mixed lymphocyte cultures (MLC), inhibited stimulation of B10.A splenic responders by both B10 and B10.D2/nSn irradiated, splenic stimulators. The B10 stimulation was suppressed approximately 85% whereas the mean suppression of B10.D2 stimulation was approximately 60%. In the ofrmer case, the serum contained antibodies reactive with multiple major histocompatibility complex determinants on the stimulator cells. In the latter case, the cytoxic reactivity of the serum was directed principally against an I region-associated determinant Ia.8) shared by B10 and B10.D2 and coded for by a gene(s) in the I-A subregion. The magnitude of the suppression of the response to B10.D2 cells (60%) was similar to the reduction in stimulation observed when the Ia.8 difference was eliminated genetically by using (B10 X B10.A)F1 responder cells against irradiated B10.D2 stimulators. Ihhibition of MLC by this antiserum was a function of reactivity with stimulator and not responder cells. Although some pools of F1 anti-B10 antiserum produced partial inhibition of the responder cell in a B10.D2 vs B10.Ax MLC combination, the results were inconsistent and not correlated with the anti-Ia.8 cytotoxicity titers. In addition, an F1 anti-B10 antiserum pool, which consistently failed to inhibit the responder cell, nevertheless inhibited both irradiated B10.D2 and (B10.A X B10.D2)F1 cells from stimulating B10.A responder cells. However, this same antiserum did not inhibit stimulation of B10.D2 responder cells by the (B10.A X B10.D2)F1 stimulators. Thus, the binding of antibodies to the non-stimulating antigens on the F1 stimulator cell did not interfere with the capacity of the appropriate stimulating antigens to cause stimulation. All of these results are consistent with the hypothesis that Ia allo-antigens are the major stimulating determinants of I region-associated MLC reactions.


Motheaten, an immunodeficient mutant of the mouse. II. Depressed immune competence and elevated serum immunoglobulins. Mice homozygous for the recessive mutation motheaten (me) are deficient in capacity for immune response but show an elevated level of serum immunoglobulins. In comparison to spleen cells from normal sibs, spleen cells from me/me mice have a severely depressed 19S PFC response to SRBC. In the GVH assay, spleen and thymus cells from motheaten donors caused significantly weaker reactions than like cells from normal sibs. Serum electrophoretic patterns of motheaten mice showed increased levels of alpha-, beta-, and gamma-globulins and decreased levels of albumin. Increases in quantities of all major classes of immunoglobulins were found in serum of me/me mice 5 weeks of age and older. Elevation of serum IgM was evident by 3 weeks of age and had reached 25 times the levels in normal sibs by 6 weeks of age. Immunoelectrophoresis and Ouchterlony analysis showed motheaten serum to have both  kappa and lambda2 light chains. Evidence of autoimmunity was found in motheaten mice in the granular deposition of IgM and IgG in kidney glomeruli. Motheaten mice, thus, appear to have a severe immune deficiency, but the basic nature of the deficiency is not yet known.


Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis. Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.


Genetic control of the immune response to nuclease. II. Detection of idiotypic determinants by the inhibition of antibody-mediated nuclease inactivation. The humoral response of mice to staphylococcal nuclease has previously been shown to be controlled genetically by H-2-linked Ir gene(s). In order to examine the possible contributions of variable region immunoglobulin genes to this genetic control, we have developed a system for the detection of idiotypic determinants on anti-nuclease immunoglobulin molecules. Antisera to nuclease were raised in two high responder strains, A/J and SJL. The corresponding antibodies were purified by affinity chromotography on Sepharose-nuclease columns, and were used to immunize groups of Lewis rats. An assay system was developed to assess the inhibition of antibody-mediated inactivation of nuclease activity by the rat antisera thus produced. Despite the presence of many species-specific anti-mouse immunoglobulin antibodies in these sera, inhibition of antibody-mediated enzyme inactivation was found to be specific for anti-nuclease antibodies of the immunizing strain. The inhibition could not be removed by extensive absorption with normal serum proteins from the antibody-producing strain, and was shown to require antibodies directed toward binding sites of the anti-nuclease antibodies. This inhibition thus defines idiotypic determinants of anti-nuclease antibodies.


Antibodies specific to isoniazid and isonicotinic acid. Rabbit antisera to isoniazid (INH) and its major metabolite, isonicotinic acid (INA), were prepared by immunization with conjugates of these compounds with human serum albumin. The antisera were rendered hapten-specific by exhaustive absorption with the immunizing carrier. Purified anti-hapten antibodies were also isolated with appropriate immunosorbents. As demonstrated by inhibition of the quantitative precipitin curves and of precipitating immune complexes in immunodiffusion tests, the antibodies to the two haptens reacted with either INH or INA, and also with isonicotinamide (INC); these three related molecules share the isonicotinyl group. The relative effectiveness of inhibition by free hapten of precipitating immune complexes consisting of either anti-INH or anti-INA antibodies and the related hapten-protein conjugates was INH greater than INC greater than INA.


Reconstitution of the somatic (O-) antigenic scheme for Providencia and preparation of O-typing antisera. The somatic (O-) antigens of the type strains of the providencia antigenic scheme were examined for their biochemical reactions and their O-specificities. The scheme of 62 O-antigens was reconstituted from 52 original type strains and 10 strains substituted for originals that either were biochemically atypical of the genus or showed inappropriate serological reactions. Thirty-six type strains showed no significant relations with other type strains, and antisera could be used for typing without absorption. Among 26 type strains, significant reciprocal relations were demonstrated, and each cross-reacting antigen was examined for specificity and for its distribution among the type strains. Antisera to these strains required absorption with cell suspensions of other type strains for production of specificity in O-typing. Each typing antiserum, at low dilution, was shown to agglutinate homologous, but not heterologous, cell suspensions of type strains, and this result demonstrated the required specificity for typing on the basis of the O-antigens.


Relation of e antigen to hepatitis B virus infection in an area of hyperendemicity. A study of the e determinant of hepatitis B surface antigen in an area of hepatitis B hyperendemicity revealed that the presence of e antigen or of antibody to e in the sera of individuals was specifically related to evidence of past or present infection with hepatitis B virus. Among asymptomatic long-term carriers of hepatitis B surface antigen, presence of the e antigen was associated with elevated levels of aspartate and alanine aminotransferases in serum; this observation suggested that the e antigen might be a marker for persisting hepatic dysfunction. Higher levels of DNA polymerase found in carriers of the surface antigen with e antigen suggested that these individuals might have a higher level of circulating Dane particles and thus, perhaps, a higher level of hepatitis B virus infectivity.


Retention of lipolytic products in chylomicrons incubated with lipoprotein lipase: electron microscope study. Early effects of lipolysis on the structure of chylomicrons in vitro were studied in rat chylomicrons incubated with purified bovine mild lipoprotein lipase at pH 8.1. The amount of the albumin added to the incubation medium was limited so that free fatty acids (FFA) and partial glycerides formed during lipolysis would accumulate in the chylomicrons. The structures visualized in lipolyzed chylomicrons was found to be affected by pH during preparation of specimens for microscopy, whether fixed with OsO4 and sectioned, or stained with sodium phosphotungstate and examined as whole mounts. Circular aqueous spaces were present in the triglyceride core of lipolyzed chylomicrons processed at pH 8.1 and 7.4. Sometimes the spaces contained aggregates of osmiophilic material and whorls of bilayered lamellae. The spaces were replaced by lamellar structures having a periodicity of 40 A, in chylomicrons processed at pH 5.5, and the spaces and lamellae were both absent at pH 3.0. The findings indicate that these spaces were lined by a lipid monolayer which formed bilayered lamellae under certain conditions. It is concluded that the monolayer lining the aqueous spaces is an inward extension of the chylomicron surface film produced by the accumulation and movement of lipolytic products, FFA and partial glycerides, in the interfacial plane between core triglyceride and water.


Irradiation treatment of inoperable squamous cell carcinomas of the oral cavity and oropharynx after partial synchronisation. Bleomycin has proved to be an efficacious cyclus-synchronising drug in the sense that it arrests the radiosensitive G2-phase directly. In comparison with 5-Fluor-uracil, Bleomycin possesses a more effective cell-kinetics. An analytical clinical remission with the application of Bleomycin, coupled with a higher incidence of local and general side effects.


Effect of obesity on endogenous fibrinolytic activity in diabetes mellitus. Examination of the fibrinolytic system of 221 diabetics with varying grades of under- and overweight revealed not only an elevated fibrinogen level and a significantly decreases spontaneous and stimulated fibrinolytic activity in obesity, but also a highly significantly decreased activity of plasminogen activator of the vessel walls in these patients. Similar, but less marked, changes were found in obese non-diabetics. Thes changes imply a decreased ability to remove fibrin deposits within the lumina of small and large vessels and thus an increased risk of thrombosis, and they may be closely related to the high frequency of late complications in diabetes mellitus.


The serological classification of Neisseria gonorrhoeae. I. Isolation of the outer membrane complex responsible for serotypic specificity. Neisseria gonorrhoeae has been subdivided into several classes of serological distinct groups. The serotyping system is based upon the antigenic specificity of a protein serotype antigen. This protein is the major polypeptide of the outer membrane of the gonococcus and accounts for over 60% of that membrane's total protein. The serotype antigen complex was isolated by mild extraction of intact organisms in 200 mM lithium acetate buffer, pH 6.0 with 10 mM EDTA for 2 h at 45 degrees C. The extract was fractionated on Sepharose 6B and partially purified by precipitation at pH 4.2 by addition of 10% (vol/vol) acetic acid. Each serotype antigen has a unique subunit molecular size as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Preliminary typing of a gonococcal strain may be performed by comparative SDA-PAGE. To date, 16 different serotypes, representing a diverse distribution, have been isolated.


Requirement of precommitted cells as targets for the augmentation of lymphocyte proliferation by leukocyte dialysates. After our initial report tha leukocyte dialysates containing transfer factor augment the thymidine incorporation of antigen-stimulated lymphocytes, we have adapted the system to microleukocyte cultures. This modification permits both (a) the simultaneous assay of a single dialysate on the cells of multiple individuals, and (b) the assay of multiple dialysates on the cells of a single individual. The data thus secured, demonstrate that dialysates from both skin-test-positive and -negative donors produced similar degrees of augmentation whether the data are expressed as an arithmetic difference or as a ratio. When expressed as an arithmetic difference, the amount of augmentation is increased in proportion to the level of thymidine incorporation of the assay cells when they were stimulated by antigen alone. When expressed as a ratio, however, the degree of augmentation is independent of the response of the assay cells. An analysis of the ability of dialysates to engage previously uncommitted lymphocytes and thus to augment thymidine incorporation, revealed that precommitted cells were required. In these experiments, antigen-reactive cells were deleted from populations of peripheral blood lymphocytes by incubation with purified protein derivative of tuberculin, diphtheria toxoid, or streptokinase-streptodornase in the presence of [3H]thymidine of high specific activity. This deletion depressed or abolished the effect of dialysate on the residual population when it was recultured with the same antigen, but the effect on the response of the remaining lymphocytes to other antigens was unaltered. In this study, leukocyte dialysate appeared to augment nonspecifically the thymidine incorporation of an antigen-specific precommitted clone of lymphocytes. The relationship of these adjuvant effects on peripheral blood lymphocytes in vitro to the specific and nonspecific activities of transfer factor in vivo remains to be elucidated.


Gangliosides as markers for murine lymphocyte subpopulations. Antibodies to GM1 ganglioside were used to study murine lymphocyte populations. In A, AKR, and BALB/c mice, anti-GM1 reacts with thymocytes and peripheral T cells. This reactivity of anti-GM1, studied by immunofluorescence, is independent of Thy-1 type and appears to be related to the reactivity of cross-reacting antibodies to asialo GM1 and GD1b, rather than GM1 itself. In addition, a subpopulation of lymphocytes reacting with anti-GM1 and anti-immunoglobulin has been found in approximately 26% of the peripheral lymphocytes of C3H mice, nude mice, and nude heterozygotes. This subpopulation is found in small numbers in A, AKR, and BALB/c mice. These studies demonstrate that antibodies to a chemically defined antigen can be used to identify T cells in many strains of mice and may delineate previously unrecognized lymphocyte subpopulations.


Idiotypic analysis of lymphocytes in vitro. II. Genetic control of T-helper cell responsiveness to anti-idiotypic antibody. When the IgG1 fraction of anti-idiotypic antibodies raised in guinea pigs is injected into mice, sensitization of idiotypic T and B lymphocytes occurs (1-3). In the present study we analyze the genetic requirements for T-helper cell sensitization by anti-idiotypic antibody. This was done by measuring, in a suitable panel of mouse strains, helper cell responsiveness to two anti-idiotypic reagents which recognize distinct, strain-specific idiotypes, namely the A5A and the S117 marker. Whenever helper cell sensitization by anti-idiotypic antibody was successful, helper function could be specifically inhibited by the same and only the same anti-idiotype. This indicates that helper cells induced by anti-idiotypic antibody express idiotypic determinants on their receptors for antigen. Helper cell sensitization by anti-idiotypic antibody was found in all strains expressing the corresponding or a cross-reactive idiotype at the immunoglobulin level. Idiotype-negative strains were always unresponsive to anti-idiotypic stimulation. In addition, responsiveness did not depend on the H-2 haplotype. Since the A5A and the S117 idiotype are markers for V genes in the heavy-chain linkage group, the present results support the view that the same genes in the Ig-1 complex code for variable portions of immunoglobulins and T-helper cell receptors.


X-gp70: a third molecular species of the envelope protein gp70 of murine leukemia virus, expressed on mouse lymphoid cells. Three variants of the gp70 envelope component of MuLV are now recognizable serologically: GIX-gp70, 0-gp70, and X-gp70. The last of these, X-gp70, has so far been found only in mice or cells producing abundant C-type virus. This distinguishes X-gp70, provisionally, from the GIX-gp70 and 0-gp70 variants, each of which can be expressed on normal thymocytes without accompanying virus production, as exemplified by mouse strains 129 and B6, respectively. The X-gp70 genotype, however, is not limited to strains of mice-producing abundant virus, because X-gp70+ leukemias occur in strains of mice which do not produce a great deal of virus and whose thymocytes and other tissues are X-gp70-; this is analogous to the appearance of GIX+ leukemias in GIX- mouse strains.