Matching of chemotherapy to mouse strain and lymphoid tumor type to prevent tumor-induced suppression of specific T- and B-cell functions. Specific immunological and hematopoietic functions were studied during treatment with antineoplastic agents in mice bearing syngeneic lymphoid tumors: 70Z/2, a B-cell lymphoma of C57BL X DBA/2 F1 (hereafter called (BD2F1) mice; EL4, a T-cell lymphoma of C57BL/6 mice; or J774, a macrophage tumor of BALB/c mice. Both B- and T-lymphocyte function (antibody-forming cells and cell-mediated lymphocyte lympholysis toward alloantigens) were suppressed in spleen cells of mice bearing these tumors. Other hematopoietic functions (granulocyte, macrophage, and megakaryocyte progenitor cells) were variably influenced by growth of these lymphoid tumors. J774 enhanced, but 70Z/2 suppressed, megakaryocyte progenitor cells. J774 and 70Z/2 increased levels of granulocyte-macrophage progenitor cells. EL4, the T-cell lymphoma, did not influence either cell type. Significant variation in strain sensitivity to drug toxicity and drug effectiveness in different tumor-host systems was observed. Increased median survival time with reversal of tumor-induced immune dysfunction, without toxicity to hematopoietic progenitor cells, was realized in two tumor-host-drug combinations. Polyinosinic-polycytidylic acid was effective against J774, while actinomycin D was active against 70Z/2. Mitomycin C effectively reduced tumor load, as evidenced by loss of splenic tumor colony-forming cells for all three tumors. This agent prolonged survival and concomitantly restored immunological responsiveness in hosts immunosuppressed by growth of 70Z/2 or J774. Paralleling tumor reduction with mitomycin C therapy, the splenic hematopoietic progenitor and colony-forming B-cells were reduced in tumor-bearing and tumor-free mice, thus compromising its therapeutic effectiveness. 1-beta-D-Arabinofuranosylcytosine reduced tumor load with marginal toxicity toward hematopoietic progenitor and colony-forming B-cells. However, immune responsiveness was only partially restored, and median survival was not increased. The results presented show the diversity of therapeutic drug effectiveness in increasing mean survival time and influencing other life-sustaining parameters (immunological and hematopoietic functions).


Induction, inhibition, and biological properties of aryl hydrocarbon hydroxylase in a stable human B-lymphocyte cell line, RPMI-1788. Aryl hydrocarbon hydroxylase was induced in the absence of mitogens by several compounds in a stable, human B-lymphocyte cell line (RPMI-1788). Over the dose ranges tested and on molar basis the inducers, in decreasing order of potency, were 2,3,7,8-tetrachlorodibenzo-p-dioxin, dibenz(a,h)-anthracene, 3-methylcholanthrene, benzo(a)pyrene, and 1,2-benzanthracene. Potential inducers which, paradoxically, diminished basal aryl hydrocarbon hydroxylase, included 7,12-dimethylbenzanthracene, 2,5-diphenyloxazole, and chyrsene. Induction under optimal culture conditions ensured maximal activities 3- to 4 fold above basal aryl hydrocarbon hydroxylase. The characteristics of the induced [dibenz(a,h)anthracene] and basal enzymes were found virtually identical. Both had similar pH curves (optima at 8.25) and inhibitor specificity (alpha- and beta-naphthoflavones, metyrapone, and 2-diethylaminoethyl-2,2-diphenylvalerate in decreasing potency). Induced and basal enzymes exhibited similar half-lives (41, 46 hr), apparent activation energies (16.7, 16.6 kcal/mol), temperature optima (37-38, 38-39 degrees), temperature-dependence of denaturation (range, 42-50 degrees), and apparent Km's with benzo(a)pyrene (1.8, 0.8 microM). The small difference in the apparent Km was related to enzyme concentration in the incubation rather than to the quality of the enzyme.


Rejection of adenovirus 2-transformed cell tumors and immune responsiveness in Syrian hamsters. Transplantation of adenovirus type 2-transformed cell-induced newborn tumor lines to different aged hamsters revealed that the cell-mediated host defenses responsible for tumor graft rejection matured early in the second week of life. When light microscopic examinations were performed during the course of tumor development, the primary histopathological difference between progressing tumors removed from newborn or thymectomized weanling hamsters and regressing lesions from normal weanlings was the lack of an early, mononuclear cell infiltrate in neoplasms from newborn and thymectomized hosts. These results suggest that the maturation of cellular immunity determines resistance to tumor transplantation in this system. This conclusion was supported by the in vitro detection of concanavalin A-responsive lymphocytes in spleens from tumor-resistant suckling but not tumor-susceptible neonatal hamsters. Although the incomplete seeding of thymus-dependent lymphocytes to the peripheral lymphoid tissues of newborn hamsters may partially explain the deficient concanavalin A responses of neonatal spleen cells, there appears to be an additional requirement for a radioresistant, adherent accessory cell population. These findings suggest that the development of a cell-mediated immune response is necessary for the rejection of adenovirus type 2-transformed cells and transformed cell-induced tumors and that this response requires the interaction of T-cells and accessory cell populations.


Characterization of factors stimulating differentiation of mouse myeloid leukemia cells from a Yoshida sarcoma cell line cultured in serum-free medium. A clone, YS-T22, of cells from Yoshida sarcoma cell line, YSSF-212T, grown in "serum-free" culture medium produced factors stimulating differentiation of mouse myeloid leukemia cells (M1) to macrophages and granulocytes. The formation of macrophages and granulocytes was accompanied by induction of phagocytosis, locomotive activity, and lysosomal enzyme activities. The rates of induction of these differentiated phenotypes were proportional to the concentration of the factor added and the period of treatment. The factor stimulating differentiation of M1 cells was a heat-labile, nondialyzable proteinaceous substance that was inactivated by trypsin but not by ribonuclease or glycosidases. On diethylaminoethyl cellulose chromatography, the factor stimulating differentiation of M1 cells from conditioned medium of YS-T22 cells was eluted in various fractions with or without activity of the colony-stimulating factor.


Noninvasive radioisotopic technique for detection of platelet deposition in coronary artery bypass grafts in dogs and its reduction with platelet inhibitors. At 8 and 32 hours after saphenous vein bypass graft surgery in six dogs, in vivo images of the graft were obtained with a gamma camera after intravenous injection, 2 hours postoperatively of autologous platelets labeled with indium-111. Platelet deposition in the grafts could be imaged in vivo from the scintiphotos. In vitro images of the excised heart showed saphenous vein graft uptake confirming the in vivo image. In vitro determination of radioactivity in the graft averaged 17 +/- 14 times greater than in blood and 33 +/- 26 times greater than in the lung. Under identical conditions, in eight dogs treated with dipyridamole (55 mg/day) plus aspirin (325 mg/day), the grafts appeared to have considerably less platelet deposition as estimated by imaging. In vitro determination of radioactivity in the graft averaged 5 +/- 2 times greater than in blood and 8 +/- 4 times greater than in the lung (p less than 0.01). This noninvasive technique may be a promising tool for a better understanding of the role played by platelets in the process of occlusion of saphenous vein bypass grafts in man and its prevention with platelet inhibotors.


Research related to rehabilitation.

Research related to rehabilitation. Progress in cardiac rehabilitation demands that rehabilitation efforts for the patient after myocardial infarction or aortocoronary bypass surgery be integrated into a comprehensive program of acute and ambulatory cardiac care. To permit a more rapid return of coronary patients to a normal or near-normal lifestyle and role in society, further delineation of the scientific bases for all components of rehabilitation programming and identification of both barriers to and facilitators of rehabilitation are necessary to improve rehabilitative services.


The response to 1,25-dihydroxycholecalciferol and to dihydrotachysterol in adult-onset hypophosphataemic osteomalacia. The biochemical changes observed in a patient with adult-onset hypophosphataemic osteomalacia after three weeks treatment with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) followed by dihydrotachysterol (DHT) are reported. The treatment with 1,25-(OH)2D3 resulted in restoration of intestinal phosphate absorption to normal with a small rise in plasma phosphate concentration; there was no significant change in tubular reabsorption of phosphate. The tubular reabsorption of bicarbonate, which was initially low, returned almost into the normal range with normalisation of plasma bicarbonate concentration. Aminoaciduria decreased. There were no changes in plasma or urinary calcium but immunoreactive parathyroid hormone (i-PTH) which was initially elevated fell but still remained above the normal range. These changes were maintained after replacing the 1,25-(OH)2D3 treatment with dihydrotachysterol (DHT).


Pregnancy-specific beta 1-glycoprotein (SP1) determined by means of electroimmunoassay, radial immunodiffusion and nephelometry. A comparison has been made between rocket-immunoelectrophoresis (RIE), radial immunodiffusion (RID) and automated immunonephelometry (AIP) in the assay of pregnancy-specific beta 1-glycoprotein (SP1) in serum from pregnant women. Using RIE an interaction was demonstrated between the various SP1-reactive molecular populations causing a bias of up to 10%. An interaction corresponding to this phenomenon cannot be demonstrated when using RID and AIP. When correlating the serum-SP1 concentration of samples containing various ratios of SP1-reactive molecules by means of RIE, RID and AIP, it was demonstrated that there was no correlation between the results achieved using one method compared to the results achieved by either of the other methods. The results achieved using one method can therefore exclusively be judged from reference values determined using the same method. The analysis time is essentially shorter with AIP than with RIE and RID.


Occurrence of antibodies to single-stranded DNA in ANA negative patients. Five patients with clinical features of a connective tissue disease most suggestive of systemic lupus erythematosus were found to have a negative ANA by conventional screening at 1/20 dilution on mouse liver. However, significant titres of antibodies to single-stranded DNA were detected using a double antibody radioimmunoassay. Thus, certain patients have antibodies to DNA restricted to single-stranded determinants as detected by radioimmunoassay with single-stranded DNA as antigen and a negative ANA test.


Pre-B and B cells in children on leukaemia remission maintenance treatment. The percentage of pre-B cells, mature B cells and IgM plasma cells were reduced in the marrow of children receiving continuous cytotoxic drug treatment to maintain leukaemia remission, compared with children receiving intermittent drug treatment in the UKALL V trial or untreated controls. When treatment was ended, the proportion of marrow pre-B cells rose above that of the controls and remained elevated for more than 6 months. These observations define more precisely the cellular basis of suppression of antibody immunity by cytotoxic drugs. They also suggest the existence of a complex feedback control of pre-B cell numbers and B cell differentiation during recovery.


Studies on the nature of heat-labile anti-complementary activity in normal human serum. Heat-labile anti-complementary activity (ACA) appears in normal human serum during storage or heating as endogenous haemolytic activity disappears. Following gel filtration of unheated serum, two peaks of heat-labile ACA are present. The ACA of both whole and fractionated serum has previously been attributed to the presence of heat-labile immunoglobulin aggregates or immune complexes. Our data demonstrate that the heavy peak of ACA obtained by gel filtration does not bind to 125I-C1q or to Raji cells, and that its effect is abolished to C1INH, suggesting that it represents C1 rather than immunoglobulin aggregates or immune complexes. The lighter peak of ACA in fractionated serum has the functional characteristics of C1s and free C1s is demonstrable in fractions containing this activity. The ACA of whole serum likewise has functional characteristics of C1. The anti-complementary effect of C1 on guinea-pig complement would not be evident in the complement fixation assay until most endogenous haemolytic activity in human serum has been inactivated, either by heat or by storage. C1INH only partially inhibits this ACA in  serum or in solutions containing isolated C1 in high concentrations. These observations indicate that heat-labile ACA in whole or fractionated sera is due to the presence of C1 and C1s and that this activity cannot be taken as evidence for the presence of immune complexes.


Further characterization of a human T lymphocyte associated antigen. A human T lymphocyte associated antigen (TLAA), defined with appropriately absorbed antisera to the lymphoblastoid cell line HSB, was originally characterized as a 150,000 mol. wt antigen using sodium deoxycholate solubilized, 125I Bolton-Hunter labelled membranes of HSB, thymus and peripheral blood lymphocytes. The TLAA, which appears to be the major membrane protein labelled with either the Bolton-Hunter, or galactose oxidase-sodium 3H borohydride methods, is weakly labelled in vitro with a 3H amino acid mixture and appears to be a minor membrane component by Coomassie blue staining of detergent solubilized HSB membranes. The antigen is trypsin-sensitive and not extractable with 3 M KCl. The glycoprotein nature of the antigen is suggested by its binding to a Lens culinaris column and its labelling by the galactose oxidase-sodium 3H borohydride and 3H glucosamine methods. Both gel filtration chromatography and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis studies on 4% SDS gels indicate that the mol. wt. of the antigen is approximately 170,000 Daltons.


Difference in the cell proliferation and colony-forming ability of normal human T lymphocytes. Cell transfer experiments were carried out with phytohaemagglutinin-induced normal human T lymphocyte colonies after 2--10 days of primary colony growth. The cells gave a cloning efficiency of 15% after 2 days of incubation and this decreased to 0.2% with the progressive growth of the colonies. The primary colonies had a proliferative capacity to give about 240 cells after 10 days of incubation, but only contained 0.5 to one cell per colony that could form a new colony. This number of colony-forming cells per colony did not change with an increase in colony size. These results indicate that colonies maintained the same number of colony-forming cells that they started with and that the other cells could proliferate but not form colonies. It is suggested that this ability to distinguish between colony-forming and proliferative T cells may be useful for determining specific deficiencie in either cell type in various diseases.


Significance of the negative exercise test in evaluation of patients with chest pain. Fifty-three patients with chest pain and a negative exercise test at greater than 85% predicted maximal heart rate underwent coronary arteriography. Twenty-one patients (40%) had significant luminal narrowing in one or two vessels. No patient had left main disease. Pathologic electrocardiographic Q waves were present in only coronary heart disease patients (p less than 0.001). There was no difference (p greater than 0.05) in prevalence of T wave abnormalities, chest pain or ventricular beats during exercise in patients with or without coronary disease. Analysis of sex distribution revealed that typical angina pectoris was uncommon in the women (p less than 0.001) and all twenty-one coronary patients were men (p less than 0.001). We conclude that in patients with chest pain and a negative exercise test, three vessel or left main coronary artery disease is unlikely. Also, women with atypical chest pain and a negative exercise test are unlikely to have a fixed coronary obstruction.


Regional effects of propranolol on intraventricular conduction in coronary artery disease. In canines, propranolol slows conduction in acutely ischemic, but not in normal tissues. To determine propranolol effects on conduction in patients with coronary artery disease, we studied 7 patients after left anterior descending coronary artery bypass graft surgery. Bipolar electrodes were placed in the atrium, left ventricle (in the left anterior descending distribution), and right ventricle. On postoperative day 7, 3 mg propranolol were given intravenously. At a constant artrially paced rate, conduction intervals were measured from the earliest onset of the QRS in 3 simultaneously recorded surface electrocardiogram (ECG) leads to the major deflection of the electrogram recorded from each ventricle. Ten minutes after injection, conduction in the left ventricle was slowed by 4 +/- 0.3 msec (10 +/- 0.9%) and in the right ventricle by 0.4 +/- 0.3 msec (1 +/- 0.9%). QRS duration changed -1 msec (-0.8%). Stimulus to Q, a measure of propranol effect on A-V conduction, changed 16 +/- 2%. The difference in propranolol effects on left and right ventricles was significant (p less than 0.001). We suggest that in patients with coronary artery disease (1) propranolol has local anesthetic effects in slowing conduction; (2) the effects of propranolol vary with the region of ventricular myocardium; and (3) propranolol slows conduction more in the left than right ventricle. This difference may be due to potentiation of drug effects in left ventricular tissue that is abnormal due to chronic coronary artery disease.


Left main coronary artery stenosis in young patients. Among 1,041 patients less than 45 years old who underwent coronary arteriography from 1972 to 1977, left main coronary stenosis greater than or equal to 50 percent was present in 31 men (3.4 percent) and in 10 women (7.2 percent, P less than 0.05). The degree of stenosis did not correlate with the duration of symptoms, the severity of angina, the presence of a previous myocardial infarction, nor with the number of risk factors. The clinical and angiographic features in young men did not seem to differ from those described in unselected populations; however, in young women, left main coronary stenosis was often an isolated lesion associated with a short duration of symptoms, a high prevalence of hypertension, no previous myocardial infarction, and a normal ventriculogram, suggesting the possibility that a different pathophysiologic mechanism might be involved. Two deaths occurred at angiography (4.9 percent). Thirty patients underwent coronary artery bypass surgery, with one operative death and one late death; good functional results were obtained, and 21 out of 28 survivors (75 percent) were asymptomatic after a mean follow-up of 29 months.


[Urokinase treatment of phlegmasia coerulea dolens (author's transl)]. Fibrinolysis treatment with urokinase was successfully undertaken in two patients, aged 71 and 76 years, with phlegmasia coerulea dolens. In the first case, with necrosis in the fore-foot, there was significant regression of the necrotic area, but a later limited amputation was still necessary. In the second, with severe heart failure, recurrent pulmonary emboli and hyperosmolar uncontrolled diabetes mellitus, complete healing was achieved. Venous thrombectomy was not possible in these two patients because of the duration of the thrombosis in the veins of the pelvic region, necrosis had already occurred, and the patients' general condition was so serious. The advanced age and arteriosclerotic changes argued against streptokinase treatment. Mean urokinase maintenance dosage of 1000-1500 IU/kg X h, with simultaneous administration of heparin at about 20 U/kg X h, produced no significant side-effects. Minor gastro-intestinal bleeding did not require stoppage of urokinase administration.


Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the tongue, esophagus, and stomach of the adult male mouse. Epidermal growth factor (EGF) previously isolated from the submandibular gland of mice was injected ip at different circadian phases into separate subgroups of adult male CD2F1 mice. Subsequent to each of the five time points of injection (0900, 1500, 1800, 2100, and 0300 h for animals standardized to 12 h of light alternating with 12 h of darkness), five animals were killed, 4, 8, and 12 h after the injection of EGF; comparable control groups were injected only with the carrier substance. Thirty minutes before sacrifice, each mouse was injected ip with 24 muCi [3H]thymidine. Incorporation of [3H]-thymidine into the DNA of the tongue, esophagus, and stomach was determined. The results demonstrate for the first time that EGF has a strong in vivo stimulatory effect on DNA synthesis in the tongue, esophagus, and stomach (studies on other areas of the gut have not yet been completed). Under the conditions of the study, stimulatory effects occurred as soon as 4 h subsequent to injection; however, maximal stimulation occurred for all three tissues 8 h after injection. Twelve hours after injection, the levels of DNA synthesis in all tissues were generally returning to normal levels found in the control animals. The results suggest circadian variation in susceptibility to EGF in the different tissues.


The effect of 12-O-tetradecanoyl-phorbol 13-acetate on the ribonuclease activity of circulating human lymphocytes. 12-O-Tetradecanoyl-phorbol 13-acetate is a very effective tumor promotor and inflammatory agent and can act as a mitogen for a subset of T lymphocytes. We report here that even short exposure of lymphocytes to 12-O-tetradecanoyl-phorbol 13-acetate changes the balance between the levels of neutral ribonuclease and ribonuclease inhibitor. The most dramatic change occurs in a B-lymphocyte-enriched population. We find that most, if not all, of the neutral ribonuclease activity in circulating lymphocytes is associated with this population and that this activity is lost with exposure to 12-O-tetradecanoyl-phorbol 13-acetate. Both 12-O-tetradecanoyl-phorbol 13-acetate and phytohaemagglutinin increase the level of ribonuclease inhibitor in T cells. However, phytohaemagglutinin has no effect on the ribonuclease or inhibitor level of the B-cell-enriched population.


The physical performances of a single slice positron tomographic system and preliminary results in a clinical environment. Metabolic phenomena can be studied and measured non-invasively using positron emitting radionuclides and a suitably adapted tomographic system. The choice of a single slice ring camera is justified by its physical performance, which is presented here and discussed. A series of measurements with geometrical phantoms and analytical simulations have been performed to determine the critical characteristics of the system. This has permitted optimization of certain parameters enabling very interesting clinical results to be obtained at SHFJ, particularly in the area of cerebral physiopathology. In addition, the potential of obtaining absolute quantitative values of regional activity is presented. The calibration of the regional activity is presented. The calibration of the system, spatial non-stationarities, and attenuation correction, which represent the main sources of error, are considered in detail. A precision of the order of 10% should be obtainable. Such a quantitation method has been successfully applied to the in vivo study of the regional extraction of cerebral oxygen.


Experimental measurement of impulse response and noise for an emission computed tomography system. Experimental studies have been made of the impulse response and noise characteristics of a tomographic system using a gamma camera. Fourier transform, deconvolution and iterative methods have been used with a CDC 6600 computer to reconstruct images from data obtained for various experimental arrangements of sources in a cylindrical phantom. It is shown that with an appropriate attenuation correction the impulse response in the reconstruction is substantially constant, independent of the position of the source in the phantom and that the reconstruction technique used is of secondary importance. The resolution obtained for the impulse response and the relative noise level throughout the 'non image' part of the reconstructions is shown for different experimental situations. The measured variance in the reconstruction of an extended uniform activity source was found to be somewhat below the theoretical value except at high count densities (above 1,000 counts per image element) where the limit of accuracy of the reconstruction is shown to be imposed by the variation in the camera sensitivity over the field of view.


Paralytic poliomyelitis in a child with agammaglobulinemia. This paper gives the clinical, immunological and virological data on a patient with agammaglobulinemia who developed paralytic poliomyelitis. The patient was a 3 year-old boy who had a typical B-cell defect without a T-cell defect. He had profound hypogammaglobulinemia and defective plasma cells and had repeated pyogenic infections which were controlled by gammaglobulin replacement therapy. At 3 years of age, he was admitted to our hospital with suspected meningitis. He had fewer, tremor and neck stiffness for 3 days and subsequently developed paralysis in his left arm and right leg. There was lymphocytosis in the cerebrospinal fluid. A non vaccine-like strain of poliovirus type 2 was isolated from the stool.


Pharmacokinetics of fenbufen in man.

Pharmacokinetics of fenbufen in man. The pharmacokinetics of fenbufen (3,4-biphenylcarbonyl proprionic acid) a new antiinflammatory agent, and its metabolites, gamma-hydroxy-4-biphenylbutyric acid and 4-biphenylacetic acid have been studied after oral administration to seven patients with rheumatoid arthritis. Fenbufen was administered as a single oral dose of 600 mg in hard gelatine capsules. A specific, sensitive gas chromatographic method was used to measure the concentration of the three compounds. A linear two-compartment open model appeared suitable to describe the course of the plasma level of fenbufen with time. This compound appeared in the blood after a lag time of 0.45 h and the peak plasma concentration of 5.97 micrograms/ml was observed after 1.19 h. The half-life of plasma disappearance was 10.26 h for fenbufen and 10.07 h and at 9.95 for metabolites II and III, respectively.


Inhibition of T cell-mediated cytolysis by 2-deoxy-D-glucose:dissociation of the inhibitory effect from glycoprotein synthesis. Previous studies have established that T cell-mediated cytolysis can be reversibly inhibited by the hexose analogue 2-deoxy-D-glucose (2-DG) by a mechanism which is apparently unrelated to energy depletion. The possibility that the inhibitory effect of 2-DG on cytolysis was linked to its known inhibitory effect on glycoprotein synthesis was therefore investigated. In contrast to the results obtained with 2-DG, no inhibition of cytolysis was observed in the presence of tunicamycin, a potent and specific inhibitor of lipid carrier-dependent protein glycosylation. Furthermore, populations of cytolytic cells which had been pretreated with doses of tunicamycin sufficient to block the incorporation of mannose (or 2-DG) into glycoproteins were still fully susceptible to inhibition by 2-DG. Other known inhibitors of viral protein glycosylation, such as glucosamine and galactosamine, inhibited cytolysis only weakly under conditions where 2-DG was highly effective. Kinetic studies revealed that the inhibitory effect of 2-DG on cytolysis could be reversed within minutes by the addition of exogenous glucose. Furthermore, suggestive evidence was obtained that inhibition cytolysis by 2-DG was linked to a parallel inhibition of effector: target cell binding. Taken together, these results strongly suggest that the inhibitory effect of 2-DG on cytolysis can be dissociated from its effect on protein glycosylation. An alternative mechanism of action of 2-DG is suggested.


Biphasic rate of synthesis of glycoconjugates, phospholipids and DNA in concanavalin A-stimulated mouse thymocytes. Involvement of cortisone-sensitive and -resistant subpopulations. The time course of the rate of labeling of membrane components (phospholipids, glycolipids and glycoproteins) and DNA was followed in concanavalin A-stimulated CBA/J mouse thymocyte cultures. Two peaks of stimulated biosynthetic activity were noted, the first at the beginning of the cultivation and the second about 25 h later. Both early and late peaks of biosynthesis of membrane components were accompanied by blast transformation and were unimpeded by suppression of DNA synthesis by hydroxyurea. Cortisone-sensitive and cortisone-resistant thymocytes were prepared by selective agglutination of the cortisone-sensitive cells with peanut agglutinin (Reisner et al. Cell. Immunol. 1976. 25: 129) or cortisone treatment of the animals. Cortisone-sensitive cells responded early, while the cortisone-resistant population gave only the late response. The autoradiographic patterns from sodium dodecyl sulfate polyacrylamide gels of [3H]fucose or [3H]galactose-labeled glycoproteins from early and late labeling cells, and cortisone-resistant cells, were compared. Late-labeling and cortisone-resistant cells gave indistinguishable patterns, but differed significantly in their patterns from early-labeling cells. It is concluded that the two peaks of biosynthetic activity during the course of concanavalin A stimulation of thymocytes are caused by two different cell populations which require different times for maximal response and react independently of one another.


Unique lectin-binding characteristics of cytotoxic T lymphocytes allowing their distinction from natural killer cells and "K" cells. Cytotoxic T lymphocytes (CTL) can be selectively depleted from in vitro of in vivo alloactivated populations of T cells on Vicia villosa lectin adsorbents through the lectin-specific interaction with the CTL-associated surface glycoprotein T 145 (Kimura, A, Wigzell, H. and Holmquist, G., J. Exp. Med. 1979. 149: 473). Results from these and other experiments have demonstrated the general applicability of this fractionation procedure in which no constraints related to antigenic specificity of the CTL have been observed. When this fractionation procedure was applied to other compartments of cytolytic cells (natural killer cells and "K" cells), no detectable impact could be seen. This differential lectin binding would appear to offer a means of dissecting the activities of CTL from other compartments of cytolytic lymphoid cells.


Studies on T lymphocyte activation. I. Requirements for the mitogen-dependent production of T cell growth factors. Conditioned media (CM) obtained from concanavalin A (Con A)-stimulated spleen cell cultures maintain mitogen-derived T cell blasts in exponential growth for indefinite periods of time. Such purified T cell blasts do not respond to Con A, and the growth-promoting activity in CM is independent of the mitogenic lectin used for its production. However, the appearance of activity in CM is Con A dose-dependent. Furthermore, the production of these T cell growth factors is independent of B cells, while it requires both T cells and nontheta-bearing, probably Ia-positive cells present in normal and nude spleens. On the other hand, the T cell blasts which are reactive to the growth factors, neither produce CM by themselves nor can they cooperate with nude spleen cells for its production, in the presence of a mitogenic lectin. Since the concentration of growth factors in CM determines the extent of T cell growth, we propose that the growth of some T cells is necessarily dependent upon the activity in CM described here and, consequently, that the basis for T cell activation is the induction of such growth factors.


Some limits to post-antigen generation of diversity: failure to detect variants in clones of hapten-specific antibody-forming cells (AFC) developing in culture from direct AFC-progenitor B cells. A search was made for variants in clones of hapten-specific antibody-forming cells (AFC) arising by stimulation of mature B cells with either thymus-independent hapten-POL (polymerized bacterial flagellin) conjugates, or the polyclonal activator lipopolysaccharides. Enriched, hapten-binding B cells or unfractionated spleen cells were cultivated for 3-4 days at limiting dilution in the presence of thymus filler cells, and the AFC in each microculture well were then assayed for plaque formation on various hapten-sheep red cell monolayers. No variants were found from (4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP) to 2,4-dinitrophenyl specificity, nor from fluorescein (FLU) to NIP specificity. No variants were found in avidity for FLU hapten. All 374 clones examined, including clones of up to 300 AFC, appeared to be homogeneous in antibody specificity and plaque morphology under our conditions. These results differ from published findings using erythrocytes as antigens. Reasons for this discrepancy are discussed, including differences in sensitivity differences in immunological similarity between the test antigens, and in the particular B cell subsets involved.


Effects of ergot drugs on serotonergic function: behavior and neurochemistry. Several new ergot drugs were tested for behavioral and neurochemical effects related to serotonergic function. Lergotrile and bromocriptine potentiated the so-called "5-HT syndrome", a set of behaviours associated with increased serotonergic neurotransmission consequent to monoamine oxidase inhibition and tryptophan loading. Metergoline antagonized this behavior. In studies of receptor binding using 3H-5-HT or 3H-LSD, metergoline was the most potent at displacing specific ligand binding. Since the ergots also affect dopaminergic function, these results are discussed for their information on both dopaminergic and serotonergic actions of these drugs and their implications for clinical use of ergots.


Dopamine-mediated circling behaviour does not involve the nigro-tectal pathway. Extensive unilateral or bilateral electrolytic ablation of the rat superior colliculus failed to reduce apomorphine- or amphetamine-induced rotation in animals with a unilateral 6-hydroxydopamine lesion of one nigro-striatal dopaminergic pathway. These findings suggest that a nigro-tectal pathway does not play a crucial role in mediating the circling response caused by striatal dopamine receptor stimulation. However, electrolytic lesions of the dorsal tegmental decussation reduced apomorphine- but not amphetamine-induced rotation in such animals, perhaps by sectioning some commissural pathway between the two nigro-striatal systems.


Impaired amylase release from the parotid gland of rats treated with reserpine. Using an automated system for the analysis of amylase, the release of this enzyme was compared in superfused parotid gland segments from control and reserpine treated rats. Stimulant-evoked amylase release was delayed and of smaller magnitude in the glands of the treated animals and a reduction of the transmembrane K+ gradient caused a smaller and short lasting reduction in Ach-evoked release of amylase in the glands from these animals.


High K+ content explains the abolition of the action potential in amphibian sciatic nerve in vitro by Lathyrus sativus seed extract. The action potential in amphibian sciatic nerve in vitro has been reported to be abolished by the topical application of Lathyrus sativus seed extract. We have confirmed this effect, but find that it is probably caused by the high K+ content of such seed extracts and that organic neurotoxins are not implicated.


Experimental studies on hemopoiesis in the pronephros of Rana pipiens. Embryogenesis of hemopoietic cell populations in the pronephros of Rana pipiens was examined during embryonic and early larval development. Differential cell counts of Wright-Giemsa-stained cell suspensions demonstrated that granulopoiesis is the predominant hemopoietic activity in the pronephros, erythropoiesis accounts for a minor component of the hemopoietic activity (less than 10%), and lymphopoiesis within the organ is negligible. Microdensitometric analysis of Feulgen-DNA stained granulocyte populations in pronephroses from larvae that had received chromosomally labeled pronephric analgen transplants between 84 and 96 h of development demonstrated that hemopoiesis in this organ is dependent on colonization by an extrinsic hemopoietic stem cell. A similar analysis of pronephric hemopoiesis in larvae which had received chromosomally labeled, presumptive ventral blood island transplants between 62 and 67 h of development, indicates that granulopoietic cells are not derived from the embryonic blood islands. It is proposed that the pronephros may be the initial site of granulocyte differentiation during early embryogenesis. Although the embryonic origin of the hemopoietic stem cell is unknown, indirect evidence from this study indicates a dorsal stem cell compartment.


Recording psychiatric consultations: a preliminary report. Two new forms, specifically designed for computer processing of data from a contemporary consultation-liaison service, are described. The need for such data and their immediate applicability to problems currently facing this psychiatric subspecialty are discussed. Clinical, administrative, and evaluation uses are reviewed. It is hoped that this work will provide a stimulus to consultation-liason practitioners to use this system or to develop similar systems that will permit documentation, exploration, and enhancement of the consultation-liaison effort.


Effects of epidermal growth factor, fibroblast growth factor and bovine serum albumin on ornithine decarboxylase activity of procine granulosa cells. Epidermal growth factor, a potent mitrogen for granulosa cells produced a three-fold stimulation of ornithine decarboxylase activity in porcine granulose cells in vitro. Fibroblast growth factor, another compound with mitogenic activity for granulose cells, did not stimulate ornithine decarboxylase. Maximally effective concentrations of a commercial preparation of bovine serum albumin equalled the maximal effect of epidermal growth factor on this enzyme activity. The dominant stimulator(s) in the albumin preparation eluted after bovine serum albumin in gel filtration. At maximally effective concentrations, luteinizing hormone produced substantially greater stimulation than either epidermal growth factor or the bovine albumin preparation. Combinations of saturating doses of any two of these stimulators produced additive effects on enzyme activity.


Kinetics of B-lymphocytes stimulation by pokeweed Pa-1 mitogen and bacterial lipopolysaccharide. The presence of two subpopulations in mouse B lymphocytes responding to pokeweed Pa-1 mitogen or bacterial lipopolysaccharide (LPS) in terms of DNA synthesis was demonstrated. The analysis of surface markers of these two subpopulations revealed that one of the subpopulations carried the complement receptor (CR+) and the other lacked the complement receptor (CR-). The kinetics of stimulation by Pa-1 and LPS differed between these two subpopulations. CR+ -B cells exhibited the first peak of DNA synthesis 27 h after the addition of the mitogen, whereas CR- -B cells showed the first maximal response after 39 h. When the responses of CR+ -B cells 27 h and of CR- -B cells 39 h after various pulsed exposures to the mitogen were compared with those after continuous exposures to the mitogen, two successive 3 h exposures (0-3 and 12-15 h for CR+ -B cells; 0-3 and 21-24 h for CR- -B cells) yielded the same level of response as continuous exposure for 27 h (CR+ -B cells) or 39 h (CR- -B cells). These results indicate that both B-cell subpopulations require two signals for maximal stimulation.


Dependence of lymphocyte surface Ig on continuous polyclonal activation. We have previously shown that most rabbit splenic B cells cultured in a medium supplemented with 5% autologous serum require continuous polyclonal stimulation to maintain detectable amounts of surface Ig. In the absence of this stimulation B cells shed but do not replace their surface Ig. Here, we investigated the mechanism responsible for the loss or maintenance of surface Ig. We showed that the addition of inhibitors of mRNA and protein synthesis to the cell cultures completely abolished the Ig maintenance effect provided by the mitogen thereby suggesting that it did not act by 'freezing' the membrane Ig but rather by continuously stimulating resynthesis. Moreover, by labelling the surface Ig with 125I-labelled Fab anti-allotype antibody we showed that the maintenance of surface Ig by mitogen stimulation was due to the turnover of surface Ig. The cells shed and replaced their surface Ig with a half-life of about 2 h only when mitogen was present but shed without replacing the surface Ig in the absence of mitogen. Also, the B-cell mitogens, SM and LPS, were able to maintain surface Ig even at extremely small concentrations while the T-cell mitogens, Con A and PHA, failed to do so at any concentration, suggesting that direct stimulation of B cells was needed to maintain surface Ig. When spleen cells were cultured in 'crowded' conditions in the absence of mitogen they did not lose their surface Ig; under these conditions it appeared that a factor associated with the macroglobulin fraction is induced and acts in the same manner as a B-cell polyclonal activator to maintain the turnover of surface Ig. Such a factor may actually function in vivo since lymphocytes are in very close contact in the lymphoid organs. We concluded that rabbit B lymphocytes shed and replace their surface Ig with a half-life of about 2 h and that the replacement, but not the shedding of surface Ig, is dependent on continuous exogenous or endogenous polyclonal activation.


Immune activation by T-independent antigens: lack of effect of macrophage depletion on the immune response to TNP-LPS, PVP and dextran. Carrageenan, a sulphated polysaccharide, and rabbit anti-mouse macrophage serum, were used to inhibit macrophage function in BALB/c mice as well as to deplete macrophages from spleen cell cultures in an attempt to determine the requirement for macrophages in the immune response to several thymus-independent antigens. Carrageenan inhibited macrophage function and was cytotoxic at low concentrations. The ability of T and B lymphocytes to undergo mitogen-induced proliferation in the presence of PHA and PLS, respectively, was not affected by in vitro exposure of lymphoid cells to carrageenan. BALB/c mice injected with carrageenan demonstrated a suppressed immune response to SRBC, a thymus-dependent antigen, but not to E. coli LPS, polyvinyl-pyrrolidone or dextran B-1355S, all of which are known to be thymus independent antigens. The sensitivity of the in vivo immune response to SRBC after depletion of macrophages by carrageenan treatment was confirmed in vitro using the Marbrook--Diener culture system. The in vitro immune response to TNP-LPS was unaffected by either carrageenan treatment or treatment of BALB/c spleen cells with AMS and complement. The results of experiments which utilized the two anti-macrophage reagents, carrageenan and AMS, both in vivo and in vitro systems, suggest that the immune response to thymus-independent antigens does not require the participation of macrophages.


Mitogenic proteins of pokeweed. I. Purification, characterization and mitogenic activity of two proteins from pokeweed (Phytolacca octandra). Saline extracts from the roots of the pokeweed species. Phytolacca octandra were separated by ion-exchange chromatography into three fractions, Po-1, Po-2 and Po-3. Po-1 contained two monomeric proteins with molecular weights of 36,000 and 29,000 and these were partially purified by gel filtration. Po-2 was purified as a single polymeric protein composed of approximately ten 14,000 mol. wt polypeptides and is a new pokeweed mitogen. Po-3 was purified as a single polymeric protein composed of approximately four 31,000 mol. wt subunits, and apart from its polymeric structure closely resembles commercial pokeweed mitogen (PWM). Po-2 and Po-3 were mitogenic for unseparated human peripheral blood lymphocytes but the degree of mitogenic activity in Po-2 preparations was dependent on storage following purification. Purified B cells were not stimulated by either mitogen. Po-3 was a potent mitogen for T cells but preparations of Po-2 required storage before they stimulated T cells. Higher responses were observed in co-cultures of B and T cells than in separated B and T cell cultures. It is suggested that human B and T lymphocytes show synergy in their responses to Po-2 and Po-3.


Mitogenic proteins of pokeweed. II. The differentiation of human peripheral blood B lymphocytes stimulated with purified pokeweed mitogens (Po-2 and Po-6) from pokeweed, Phytolacca octandra. Purified pokeweed mitogens, Po-2 and Po-3, extracted from Phytolacca octandra, stimulated plasma cell formation in cultures of human peripheral blood lymphocytes. Plasma cell formation did not occur in cultures of purified B cells but was dependent on T-cell help. High T-cell numbers, however, suppressed plasma cell formation in mixed B- and T-cell cultures stimulated with Po-2. T-cell helper function was exerted across a major histocompatibility barrier and was not dependent on T-cell proliferation. Soluble helper factor(s) from activated T cells were not demonstrated. Po-3 was an effective B-cell stimulant only at concentrations between 0.1 and 1.0 micrograms/ml. In contrast, relatively high concentrations of Po-2 (50--100 micrograms/ml) were required to induce B-cell differentiation. More plasma cells were generated in Po-2-stimulated cultures than in Po-3-stimulated cultures and this was thought to reflect the more aggregated state of Po-2.


Contrasting effects of H-2 and Mls immunization on the polyclonal mitogenicity of murine lymphocytes. During the immune response to H-2 and Mls alloantigens, murine lymphocytes showed altered sensitivity to polyclonal mitogens. The reactivity to the T-cell mitogen PHA followed a similar pattern in both H-2 and Mls-immunized mice while the reactivity to the B-cell mitogen LPS was contrasting in the two groups. In the former group, the response exceeded control levels by the seventh day after immunization and then gradually dropped below control levels; the response of Mls-immunized lymphocytes dropped below control levels soon after immunization and remained so for the period of study. Nylon wool column-purified Mls-immunized B cells also showed a suppressed reactivity to LPS, while the T-enriched populations from Mls-immune mice when added to normal B cells lowered their LPS reactivity. Soluble factors derived from clutures of Mls-immune lymphocytes had a suppressive effect on normal B cells.


Lactate dehydrogenase isoenzyme pattern of the pre-thymic precursor cell. The thymic cell precursor was obtained from thymic rudiments of 14 day old mouse embryos of strain NMRI. The LDH isoenzyme pattern of this cell population showed a higher LDH-5 activity than the cortical and medullary thymocytes. These results indicate that during the T-cell development, a shift in the pattern occurs towards the LDH-1 end of the spectrum, as described for other murine tissues and cells. As cortical thymocytes contain less LDH-5 activity than the medullary thymocytes, however, the outcome of the latter cells from the former is in conflict with the shift toward the LDH-1 end of the spectrum. On the contrary a relationship whereby cortical and medullary thymocytes are independently derived from the thymic cell percursor, is consistent with the shift toward LDH-1.


Direct cytotoxicity against chicken erythrocytes in mice. II. Nature of immunoglobulin on cytotoxic T lymphocytes. Direct cytotoxicity raised at an early interval after immunization with chicken erythrocytes (CRBC) has been ascribed to O-positive, immunoglobulin (Ig)-bearing lymphocytes. Cytotoxicity of such effector cells was not affected by trypsinization. Cytotoxity was reduced by the treatment of effector cells with anti-IgG serum and complement or by the addition of anti-IgG serum to culture medium for cytotoxicity test. When the effector cells were treated with trypsin before cytotoxicity test, such procedures with anti-IgG serum did not affect their cytotoxicity. Ig molecules on cytotoxic T lympohcytes may not be an essential element for antigen recognition. This explanation may be supported by the fact that cytotoxicity against CRBC could be raised in 600 rad irradiation, thymus-cell transferred mice in the absence of antibody production.


Diphtheria toxin-binding glycoproteins on hamster cells: candidates for diphtheria toxin receptors. Diphtheria toxin-binding glycoproteins of high molecular weight (greater than 100,000) were identified on the surface of lymph node and thymus cells from hamsters, a diphtheria toxin-sensitive species. These diphtheria toxin-binding glycoproteins also interacted with CRM197 protein, which possesses toxin-blocking activity, but not with diphtheria toxoid, fragment A of diphtheria toxin, or cholera toxin, all of which lack toxin-blocking activity. These observations are consistent with the hypothesis that the detected diphtheria toxin-binding glycoproteins are involved in intoxication of cells by this toxin and possibly serve as the plasma membrane receptors for diphtheria toxin.


Elevated adjuvanticity of non-acid-fast mycobacteria. Administration to guinea pigs of non-acid-fast (NAF) Mycobacterium avium resulted in a sevenfold greater circulating antibody response than that for the animals receiving ovalbumin in Freund incomplete adjuvant alone. The ability of NAF M. avium on induction of delayed hypersensitivity was comparable or somewhat stronger than that of normal M. avium. The in vivo enhancement of circulating antibody response by NAF M. avium associated with a marked stimulating effect of it on proliferation of B cells in regional sites. Induction of local granuloma at the injection site of NAF M. avium was less than that induced by injection of normal M. avium. Phagocytic activity of the reticuloendothelial system in mice was also greatly increased by administration of NAF M. avium.


Mitotic activity of thymocytes in a synthetic tissue culture medium. Effect of L-alanine. DNA synthesis in guinea pig thymocytes suspended in RPMI 1640 medium increased to a peak after 4-5 h in culture and was followed by increased mitotic activity, indicating that many thymocytes in S phase proceeded through G2 into mitosis. Addition of L-alanine to the medium markedly increased the DNA synthesis within 1 h and the mitotic frequency from 6 h. The increase in DNA synthesis when L-alanine was present in the medium was thus caused by an increased number of cells in S phase. Human thymocytes cultured in RPMI 1640 for 18 h had a low mitotic frequency. Addition of L-alanine immediately started DNA synthesis in the arrested thymocytes resulting in increased mitotic activity from 6 h later. The results show that L-alanine is a growth factor for guinea pig and human thymocytes and should be included in tissue culture media used for such cells. Growth of thymocytes in vitro was partly synchronized, and the mitotic studies indicated that many cells had entered S phase near the start of incubation.


The bleaching of the bacteriorhodopsin chromophore by ionizing radiation. The visible chromophore of bacteriorhodopsin, BR(570), undergoes progressive bleaching when subjected to 60CO gamma-irradiation. The low G-value for bleaching confirms that the site of the chromophore is highly protected. Positive and negative circular dichroic (CD) bands associated with the chromosphone undergo concomitant decrease in a manner which is consistent with two independent chromophores rather than exciton coupling between neighbouring chromophoric site.


The mobility of curium-244 dioxide in the bronchially intubated rat. The mobility of curium dioxide in the rat after pulmonary intubation has been investigated by administering suspensions containing different particle size ranges of the oxide. A major factor influencing the movement of curium from lungs to blood is the formation of hydroxide or hydrous oxide particles about 0.001 micrometer in diameter. This process is sufficiently rapid for the lung clearance kinetics of the dioxide to resemble those of a soluble compound more closely than those of an insoluble one. Filtration of 0.001 micrometer particles through the kidneys results in considerably enhanced excretion of curium relative to administered curium citrate. It is concluded that current metabolic models, which assume that solubility in the lung is a prerequisite for transport in body fluids, do not adequately describe the behaviour of curium fromthe standpoint of radiological protection.


The relationship between o.e.r., r.b.e. and number of fractions for X-ray- or neutron-induced skin damage. The skin reactions in aerated and hypoxic mouse tails after single or fractionated doses of 250 kV X-rays or fast neutrons (6 MeV deuterons on beryllium) have been measured. The o.e.r. for one to sixteen fractions of X-rays remains constant, while that for one to ten fractions of neutrons decreases with increasing neutron fractionation and decreasing neutron dose/fraction. The o.e.r. for X-rays was 1.7, for single-neutron doses 1.4, and for ten fractions of neutrons 1.25. It was anticipated that the o.e.r. for neutron-induced damage would decrease further as neutron fractionation is increased because the contribution to damage from the highest LET components of dose, the alpha and heavy recoil particles, would increase relative to the lowe LET components. The r.b.e. values obtained for skin damage were higher at all neutron doses/fraction examined in this study on tails than all those previously obtained in studies on skin at other sites on four species. This may be due to the influence of hypoxia on the r.b.e. measurements in the mouse tail.


Implication of tryptophan and tyrosine in the binding of p-nitrophenyl-alpha-D-mannopyranoside by concanavalin A. A radiolytic study. Equilibrium dialysis has been used to study the effects of 60Co gamma-radiolysis on the carbohydrate binding site of Con A. Reaction of eaq- and OH. with Con A is accompanied by a decrease in the number of binding sites. Modification of the binding site by (Br)2- is independent of the initial concentration of Br-. Reaction of (SCN)2- with the protein, however, is dependent on the initial SCN- concentration. The results imply that tryptophan and possibly tyrosine are probably involved in the carbohydrate binding process.


Radiation-induced DNA strand breaks and their repair in the developing rat brain. Rats, 5, 10 or 25 days old, were 60 Co gamma irradiated. The induction of DNA strand breaks was studied after killing the rats within 1 min after irradiation, and the repair of the induced breaks after various intervals up to 180 min. Cell suspensions were prepared from the brain and samples were transferred into alkaline solutions. The fraction of DNA remaining double-stranded after 30 min alkali treatment was estimated after separation of single- and double-stranded DNA on hydroxylapatite. The amount of DNA strand breaks induced per Gray (1--8 Gray) was found to be in accordance with earlier in vivo studies of the mouse small intestine and mouse spleen. The DNA strand breaks in the rat brain induced by 4 Gray 60Co gamma irradiation were repaired 30 min after irradiation in all age groups studied.


Effectiveness of 0.3 keV carbon ultrasoft X-rays for the inactivation and mutation of cultured mammalian cells. Carbon K characteristic ultrasoft X-rays of energy 0.278 keV were found to be effective in inducing inactivation and mutation to thioguanine resistance in cultured V79 Chinese hamster cells and human diploid fibroblasts. These X-rays act as a probe of the sensitive sites within the cells since they produce low-energy photoelectron tracks of range about 7 nm; this is an order of magnitude smaller than those produced by the 1.5 keV aluminium X-rays used in previous studies. A detailed interpretation of the results requires assumptions to be made about the positions of the sensitive sites within the cells but, for any reasonable set of assumptions, the carbon X-rays are found to be more effective than gamma-rays and are probably at least as effective as long tracks of helium ions of similar LET. These observations extend the conclusions previously drawn from the observed effectiveness of aluminium X-rays regarding the sizes of the subcellular sites involved in inactivation and mutation. They imply that the sensitive sites smaller than about 7 nm, and that highly localized energy depositions consisting of less than or approximately 14 ionizations are sufficient to produce biological effects. These results are also in contradiction to models of radiation action which require relatively large sites, such as the usual form of the 'theory of dual radiation action'.


Dependence of the nucleoside effect on linear energy transfer. L929 cells were irradiated by cyclotron-produced neutrons and by 14.8 MeV monoenergetic neutrons. For comparison cells were also irradiated by 60Co gamma rays. Following irradiation cells were treated by an equimolar solution of deoxyribonucleosides, and the effect on cell survival measured. Results show that nucleoside treatment was efficient after low-LET irradiation: gamma ray survival curves were altered by deoxyribonucleosides in terms of significantly increased extrapolation numbers only, but without Do change. Cells irradiated by neutrons from either of the two sources did not respond to nucleoside treatment, and consequently their survival curves remained unaltered. These results show that the nucleoside effect does occur after low-LET irradiation, but apparently not following high-LET irradiation. Since deoxyribonucleosides as well as other cell breakdown products are released in irradiated and necrotic tumours due to massive cell destruction, such a nucleoside effect could possibly enhance the cell survival and thus effect the result of radiotherapy. Absence of the nucleoside effect in case of high-LET irradiation may therefore be an additional potential gain from neutrons in radiotherapy.


Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. I. Irradiation facilities and methods. This paper, which is the first of four covering the inactivation of clonogenic capacity and induction of mutation in cultured mammalian cells, deals briefly with the general aims of the work and describes the irradiation techniques used. Human diploid fibroblasts and V79 Chinese hamster cells were irradiated as monolayers with ions of helium, boron or nitrogen at LET's in the range 20 to 479 keV micrometer-1 in H2O. The physical aspects of the irradiation including measurement of ion energies, dosimetry and uniformity of dose and also the methods of handling large numbers of samples are described in detail. Subsequent papers will present the biological methods and results and a biophysical analysis of the data.


Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. II. Chinese hamster V79 cells. Inactivation and mutation to thioguanine-resistance of V79 hamster cells were studied after irradiation with accelerated helium, boron or nitrogen ions covering a range of linear energy transfer from 28 to 470 keV micrometers-1. For all radiation qualities a dose-dependent increase in mutant frequency was found for doses giving surviving fractions greater than about 0.20. The effectiveness per unit dose for both inactivation and mutation induction increased with the linear energy transfer of the radiation to a maximum in the range 90-200 keV micrometer-1. However, the maximum mutagenic effectiveness relative to gamma-rays was about two or more times that for inactivation. It is suggested that a proportion of the radiation-induced mutants suffer extensive genetic damage, and that some forms of this damage may be induced with high efficiency by radiations of high linear energy transfer.


Mutation and inactivation of cultured mammalian cells exposed to beams of accelerated heavy ions. III. Human diploid fibroblasts. The induction of inactivation and mutation to thioguanine-resistance in cultured human diploid fibroblasts was studied after exposure to ionising radiations with LET's in the range 20--470 keV micrometer-1. Unique r.b.e. values were obtained for inactivation and mutation induction with nine different qualities of radiation. The plot of r.b.e. verus LET gave humped curves for both endpoints; r.b.e. maxima were in the LET range 90--200 keV micrometer-1 but the maximum r.b.e. value for mutation induction was almost twice that for inactivation. The accuracy of estimates of mutation induction are discussed with regard to possible selective effects against mutants during post-irradiation growth.


Influence of seed moisture content and post-irradiation hydration temperature on the kinetics of reactivity towards oxygen or decay of oxygen-sensitive sites. The rate of development of post-irradiation oxygen-dependent damage when oxygen is available, and its rate of elimination when seeds are first post-hydrated in oxygen-free water prior to their transfer to oxygenated water, was studied in barley seeds of approximately 3 per cent, approximately 8 per cent and approximately 9 per cent moisture contents at 3 degrees C, 25 degrees C and 37 degrees C. The magnitude of oxic damage at a given dose (35 krad) decreases as the initial seed moisture content increases from approximately 3 per cent to approximately 9 per cent. Significant (P = 0.01) oxic damage is observed in seeds of all the three moisture contents at 3 degrees C and 25 degrees C; however, at 37 degrees C significant (P = 0.01) oxic damage is observed only in seeds of approximately 3 per cent and approximately 8 per cent moisture contents. The magnitude of oxic damage in seeds of a given moisture content remains unaltered following oxygenated post-hydration of seeds at 3 degrees C and 25 degrees C, but it registers a significant (P = 0.01) decrease if post-hydration in oxygenated water is carried out at 37 degrees C. The radiation-induced oxygen-sensitive (An) sites react with oxygen approximately 6 to 8 times faster as compared to their rate of decay in the absence of oxygen at both 3 degrees C and 25 degrees C; however, at 37 degrees C they react only approximately 3 to 4 times faster, in seeds of all the three moisture contents. Moreover, the initiation of the decay of An sites becomes evident much earlier in very dry (approximately 3 per cent moist) seeds than in relatively moist (approximately 8 per cent and approximately 9 per cent) seeds. It is also observed that this fraction of An sites which is capable of a very rapid rate of decay in the absence of oxygen is capable also of an even more rapid rate of reactivity towards oxygen.


Sensitivity of light-grown and dark-grown Euglena cells to gamma-irradiation. Light-grown cells which contain fully developed chloroplasts were found to be more resistant to gamma-irradiation than dark-grown cells which are devoid of chloroplasts. The radio-resistance of dark-grown cells progressively increased during light-induced development of chloroplasts and, conversely, radio-resistance of light-grown cells decreased progressively with chloroplast de-development during growth in the dark. The presence of chloroplasts seemed to play a major role in the capacity of cells to recover from radiation damage, the efficiency of cellular recovery being correlatable with the degree of chloroplast development.


Effects of neonatal thymic exposure to high doses of X-irradiation. The thymic region of neonatal Swiss mice was exposed to doses varying from 1000 R to 2000 R of X-irradiation. The animals did not show any signs of wasting syndrome up to 6 months after irradiation. At this time hyperplasia of the thymus with an associated lymphocytosis was evident in irradiated animals. Antibody production to sheep red blood cells (SRBC) was not affected. However, at 12 months post-irradiation the animals showed signs of wasting disease with a progressive increase in their numbers at 18 and 24 months of age. The percentage incidence of animals with wasting disease was dose dependent. At this stage in the majority of the animals with the disease the thymus showed varying degrees of atrophy along with splenomegaly. There were no significant differences in the number of lymphocytes but the number of granulocytes showed a substantial increase. This was more evident in animals exposed to 2000 R to the thymic region. Though one observed a lowered ability to form antibodies to bovine serum albumin (BSA) with advancing age, the thymic irradiation did not affect the immune response to BSA even in animals manifesting wasting disease. An interesting observation has been the development of a severe loss of muscle power and tone in the hind limbs in a large majority of animals.


Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations. Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei are both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brings estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells.


Effect of ionizing radiations on the life span of non-transformed human fibroblasts. We studied in vitro the influence of ionizing radiations on the life span of non-transformed HF 19 human fibroblasts. The life span of surviving clones was found to be reduced when the cells had received two or three doses of 6 Gy separated by an interval of 15 doublings. In addition, this reduction in life span was greater when the cells were older at the time of irradiation.


Ultrastructural localization of alkaline phosphatase activity in the intestine of developing Rana catesbeiana. Alkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog, Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.


Effect of catecholamines on lymphopoiesis in fetal mouse thymic explants. Fetal thymic explants (14 days of gestation) from Balb/c mice were grown in organ culture for up to 12 days and the pattern of lymphopoiesis was monitored by 125IUdR incorporation and cell yields of such lobes in the presence of adrenergic agents. It was observed that, after 3 days of cultures, phenylephrine stimulated lymphopoiesis in thymic explants, whereas some inhibition was observed with isoproterenol. These observations were confirmed by autoradiography. It was also observed that cells obtained from phenylephrine-treated cultures had greater mitogen reactivity than either control (untreated) or isoproterenol-treated cultures. The possible role of cyclic nucleotides as intracellular mediators of thymocyte maturation is discussed.


Correlation of in vitro susceptibility with in vivo efficacy in mice for cefoxitin in comparison with cephalosporins. Agar minimal inhibitory concentrations and mouse protection test effective doses were determined for each of four beta-lactam antibiotics against each of 12 Gram-negative and 3 Gram-positive bacterial cultures. The beta-lactamase activity of these cultures also was studied. The data were examined to determine whether relative in vivo efficacies could be predicted from relative in vitro activities. Although such predictions were quite accurate for cefoxitin and cefazolin, this was not true for cefamandole or for cephalothin. Such poor predictability was not necessarily associated with the susceptibility of these cephalosporins to hydrolysis by bacterial beta-lactamases. Although the clinical significance of these observations is not known, these data emphasize that relative in vitro activities should be used only with caution to estimate in vivo efficacies, since not all compounds show the excellent predictability observed here for cefazolin and cefoxitin.


Translational regulation of polysome formation during dormancy of Physarum polycephalum. The translational activity of actively growing microplasmodia and dormant microsclerotia of Physarum polycephalum was investigated by analyzing the distribution of ribosomes in polysomes. Microplasmodial post-mitochondrial fractions contained substantial amounts of polysomes and ribosomal subunits but very few native monosomes. During the starvation period which preceded microsclerotium formation, polysome levels remained constant, whereas the subunit titer began to increase. During encystment ribosomal subunits continued to accumulate as the level of polysomes gradually decreased. Dormant microsclerotia contained a large surplus of stored ribosomal subunits but no detectable polysomes. However, incubation of microsclerotia with concentrations of cycloheximide sufficient to slow polypeptide elongation without affecting initiation caused the gradual reappearance of polysomes at the expense of the subunits. Under these conditions the percentage of subunits driven into polysomes reached values similar to those of actively growing microplasmodia. Microsclerotia returned to nutrient medium contained very low levels of polysomes during the lag period which preceded germination. These were formed with preexisting, stored messenger ribonucleic acid. During the germination period, polysome levels were markedly increased. This elevation was dependent on new ribonucleic acid transcription. It is concluded that dormant microsclerotia contain functional messenger ribonucleic acid and ribosomes which are subject to translational repression at the level of initiation.


Poising of the arginine pool and control of bioluminescence in Beneckea harveyi. Arginine dramatically stimulates bioluminescence in the marine bacterium Beneckea harveyi growing in minimal media, an effect that is due to increases in both the synthesis and expression of luciferase. To elucidate the mechanism of this phenomenon, studies were made of the transport and metabolism of arginine in B. harveyi. The transport of arginine and lysine involves two kinetically distinct transport systems for the uptake of arginine and lysine. In contrast, ornithine is transported only by a system common to all three amino acids. The internal amino acid pools were measured in mutants that do not show stimulation of bioluminescence by arginine and in wild-type cells that do. In minimal media, the internal arginine pools are undetectably low. Furthermore, exogenously added labeled arginine is rapidly transported and converted to citrulline and argininosuccinate. The results can be accommodated by a model in which the internal arginine is poised at a very low concentration; the stimulatory effect of exogenous arginine on luciferase biosynthesis occurs at the transcriptional level, and the actual mediator can be either arginine or argininyl transfer ribonucleic acid.


Characterization of a peptide released during the reaction of human alpha 1-antitrypsin and bovine alpha-chymotrypsin. The release of a peptide (molecular weight: about 3,600) was observed during complex formation between human alpha 1-antitrypsin (alpha 1-AT) and bovine alpha-chymotrypsin, when monitored by gel-electrophoresis in the presence of sodium lauryl sulfate. Release of the peptide was proportional to the extent of complex formation. Peptides of the same molecular weight were also released during the complex formation of alpha 1-AT with bovine trypsin or porcine elastase. The peptide released from the complex with bovine alpha-chymotrypsin was composed of 32 amino acid residues, which did not correspond to the composition of any 32 amino acid segment in the bovine alpha-chymotrypsin sequence. The N- and C-terminal sequences of the peptide were determined to be H-(Ser)-Ile-Pro-Pro-Glu- and -Gln-Lys-OH, respectively. Though there was some uncertainty as to the N-terminal sequence, it is quite different from that of the original alpha-AT molecule, and showed a similarity to the sequences of the leaving group sides of the reactive sites in some legume proteinase inhibitors. The C-terminal 2 residues were identical with those of native alpha 1-AT. These results suggest that the peptide was released from the C-terminal region of alpha 1-AT uon interaction with alpha-chymotrypsin. It is tempting to suggest that alpha 1-AT inhibits a serine proteinase by the acyl enzyme mechanism at a residue adjacent to the amino group of the N-terminus of this peptide and that this peptide is liberated as a leaving group in the enzymic process.


Isolation from lactalbumin hydrolysate of a high molecular weight mitogenic factor. A new mitogenic factor has been isolated from tissue culture grade lactalbumin hydrolysate. Incubation of postconfluent Swiss 3T3 cells in serum-free medium containing lactalbumin hydrolysate resulted in enhanced synthesis and release of plasminogen activator. Sephadex G-100 gel filtration of concentrated, dialyzed lactalbumin hydrolysate revealed two fractions, LH-FI and LH-FII. LH-FI elutes at the void volume, indicative of high molecular weight, and contains all plasminogen activator stimulatory activity of the original lactalbumin hydrolysate, whereas LH-FII has no activity. In addition, LH-FI also induces DNA synthesis in Swiss 3T3 cells in a dose-dependent fashion, 1 to 2 microgram/ml being equivalent to 10% fetal calf serum. Again, LH-FII is without effect. Induction of DNA synthesis in LH-FI or serum-stimulated quiescent sparse cells followed essentially identical kinetics at least through the first 20 h. Furthermore, LH-FI also enhances 3T3 cell growth. Preliminary results of Sepharose 4B filtration of LH-FI reveal the presence of five subfractions each with plasminogen activator stimulatory as well as mitogenic activity.


Mortality in gold and coal miners in Western Australia with special reference to lung cancer. Cohorts of 1974 gold miners and 213 coal miners in Western Australia surveyed for respiratory symptoms, smoking habits, occupational history and radiographic evidence of pneumoconiosis have been followed up for 13-14 years. Overall, neither group had a significantly higher mortality than expected from the experience of Western Australian men in general. Lung cancer mortality was relatively high in the gold miners (59 deaths observed, 40.8 expected) but weakly and inconclusively related to the extent of their underground mining experience. Cigarette smoking may explain the excess of lung cancer in the gold miners because the prevalence of the habit in the latter (66.3%) was higher than in the coal miners (58.7%) or in other men in Western Australia (53.2%). Radiographic evidence of silicosis was present in 21.7% of the gold miners but did not appear to have contributed substantially to their mortality. The coal miners showed a lower than expected rate of lung cancer but an excess of deaths from all other forms of cancer (11 observed, 5.6 expected). This excess was not attributable to any one cancer site and cannot be explained readily.


Comparison of two media for isolation of haemophilus vaginalis. Starch agar and V agar were comparable in the isolation of Haemophilus vaginalis from vaginal discharge specimens. Each medium had certain advantages over the other, which are described.


Carbohydrate of the factor VIII/von Willebrand factor in von Willebrand's disease. We have examined the plasma Factor VIII/von Willebrand factor (FVIII/vWF) molecule from 16 patients with von Willebrand's disease, and have found no evidence of a significant decrease of carbohydrate content in 15 of these patients. FVIII/vWF was isolated by preparative counter immunoelectrophoresis directly from plasma using antibody to Factor VIII-related antigen, reduced in sodium dodecyl sulfate in the presence of urea, and electrophoresed in 5% polyacrylamide gels to separate the FVIII/vWF subunit from other proteins. Duplicate gels were stained by either the periodic acid-Schiff (PAS) reaction or by Coomassie Brilliant Blue G250. The ratio of Coomassie: PAS was determined by spectrophotometric scanning of the gels. Transferrin was used as an internal reference standard. The ratio for 23 normal individuals was 2.4+/-0.38 and the observed range was 1.8-3.8. 15 patients with von Willebrand's disease fell within this range. One patient independently reported as having decreased FVIII/vWF carbohydrate was also studied by this technique. A ratio of 6.8 was found, indicative of decreased, though not absent, carbohydrate. Cold insoluble globulin did not represent a significant contaminant in these analyses. 11 of the von Willebrand's disease patients with normal FVIII/vWF carbohydrate had abnormal crossed immunoelectrophoretic patterns characterized by absence of the less anodic forms of Factor VIII-related antigen. Four patients had normal patterns. These studies indicate that an absence or decrease of PAS reactive FVIII/vWF carbohydrate is not a consistent abnormality in von Willebrand's disease.


In vitro studies of poison oak immunity. I. In vitro reaction of human lymphocytes to urushiol. Poison oak, ivy, and sumac dermatitis is a T-cell-mediated reaction against urushiol, the oil found in the leaf of the plants. This hapten is extremely lipophilic and concentrates in cell membranes. A blastogenesis assay employing peripheral blood lymphocytes obtained from humans sensitized to urushiol is described. The reactivity appears 1--3 wk after exposure and persists from 6 wk to 2 mon. The dose-response range is narrow, with inhibition occurring at higher antigen concentrations. Urushiol introduced into the in vitro culture on autologous lymphocytes, erythrocytes and heterologous erythrocytes produces equal results as measured by the optimal urushiol dose, the intensity of reaction, and the frequency of positive reactors. This suggests that the urushiol is passed from introducer to some other presenter cell. Although the blastogenically reactive cell is a T cell, there is also a requirement for an accessory cell, found in the non-T-cell population, for reactivity. Evidence is presented that this cell is a macrophage.


In vitro studies of poison oak immunity. II. Effect of urushiol analogues on the human in vitro response. Studies were performed to ascertain the effect of urushiol analogues on the in vitro lymphocyte blastogenesis elicited by urushiol in peripheral blood lymphocytes taken from individuals sensitized to poison oak or ivy. Urushiol is a mixture of alkylcatechols composed of a catechol ring coupled to mono-, di-, or tri-unsaturated C-15 or C-17 carbon side chains. Each of these two moieties, catechol ring and side chain, was tested for its role in eliciting reactivity. Analogues tested represented the catechol ring (3-methylcatechol), the mono- or di-unsaturated side chain (oleic or linoleic acid), and the saturated side chain coupled to a catechol ring (pentadecylcatechol), a blocked catechol ring (heptadecylveratrole), or a resorcinol (pentadecylresorcinol). Urushiol with a blocked catechol ring (urushiol dimethyl ether) was also included. Of these, only pentadecylcatechol evoked reactivity in sensitized lymphocytes, and this reactivity was only a fraction of that evoked by urushiol. This suggested that the system has some requirement for the side chain, and that the catechol ring is critical for reactivity. This was further investigated by testing the ability of some of these analogues to inhibit urushiol-specific blastogenesis. No inhibition was noted with compounds bearing the saturated side chain with modified ring structures (pentadecylresorcinol and heptadecylveratrole). However, both 3-methylcatechol and pentadecylcatechol (at equimolar concentrations) blocked reactivity. The results of our experiments suggested that although both the side chain and the catechol ring are required for reactivity, the latter is most critical. Unsaturation in the side chain is important for maximal reactivity because the saturated catechols were only partially as active as the urushiol oil. There may be a greater dose requirement for the catechol ring than for the side chain.


T-lymphocyte differentiation in vitro in severe combined immunodeficiency. Defects of stem cells. A study of T-lymphocyte differentiation was made on fractionated bone marrow cells from normal volunteers and from 11 patients with severe combined immunodeficiency (SCID) using normal thymic epithelial monolayers and their culture supernates as inducing agents. Normal marrow cells could regularly be induced to bear the human T-lymphocyte antigen (HTLA), to form rosettes with sheep erythrocytes (E rosettes), and to respond to the mitogen concanavalin A (Con A) after coculture with the thymic epithelial monolayers or their culture supernates. In contrast, studies of T-cell differentiation on the marrow cells of patients with SCID revealed varying defects, ranging from a complete "absence" of definable T-cell precursors to partial differentiation resulting in acquisition of one (HTLA) or two (HTLA and E rosettes) markers for T lymphocytes. Only in one patient was there induction of all three T-cell markers, namely, HTLA, E rosettes, and responsiveness to Con A. These observations indicate that SCID is a heterogeneous disorder in which defects of differentiation can occur at one or more multiple sites of differentiation leading the the clinical expression of T- and B-cell dysfunction. Further, our studies indicate that in T-cell differentiation, HTLA probably appears before the capacity to form E-rosettes, and development of the latter capacity is followed by a state of responsiveness to mitogens. A scheme of normal differentiation along with the defects of precursor T cells seen in SCID is presented.


Lactoferrin acts on Ia-like antigen-positive subpopulations of human monocytes to inhibit production of colony stimulatory activity in vitro. The relationship between Ia-like antigens (Ia-antigens) on human monocytes and the ability of lactoferrin (LF) to inhibit the production of colony stimulatory activity (CSA) for granulocyte and macrophage colony formation was investigated. Complement-dependent cytotoxicity of human monocytes by antiserum to Ia-antigen-reduced CSA production by 50%. LF decreased CSA production by monocytes but had no influence on monocytes insensitive to anti-Ia and complement. Anti-Ia in the absence of complement had no effect on production of CSA but blocked the inhibitory action of LF. This suggsts that LF inhibits production of CSA from an Ia-antigen-positive subpopulation of human blood monocytes. This may be of relevance to the regulation of myelopoiesis.


Effect of tobacco and nicotine on growth of Haemophilus influenzae in vitro. Two nutritionally poor bacteriological media, prepared from phosphate-buffered saline and from bronchial secretions, support the growth of Haemophilus influenzae only poorly. The presence of tobacco or pure nicotine in these media stimulates the growth of the organism, and the component(s) responsible appear(s) to be volatile.


Use of modified X-Chrom for relief of light dazzlement and color blindness of a rod monochromat. A young female rod monochromat is treated optometrically with binocular contact lenses to relieve light dazzlement and a monocular lens to improve hue discrimination. A comparative study of various absorption contact lenses results in treatment of both problems with modified X-Chrom. A hypothesis is offered to explain the physiological aspects of relief. This paper also describes her visual experiences from an early age, her signs and symptoms of typical rod monochromacy and the methods of diagnosis. It is a brief historical account comprised of theories and references of contributors to scientific information.


The problem-oriented optometric record.

The problem-oriented optometric record. Today's busy practitioner often has little time to fully review his patient's records and thereby runs the risk of not seeing all the previously collected information. The problem-oriented system of patient record keeping provides one systematic logical method for recording data with previous information easily obtained to allow the practitioner to better utilize past and present data in making patient care decisions. This paper outlines the problem-oriented method and how it may be used in daily optometric practice.


Role of self carriers in the immune response and tolerance. IV. Active T cell suppression in the maintenance of B cell tolerance to a "T-independent" antigen. Previous studies indicated that T cells are required for tolerance induction by hapten-modified syngeneic spleen cells (TNP-SC) in vivo. The role of T cells in the maintenance of this unresponsive state has been examined herein. By three criteria--limiting dilution precursor analysis, removal of T cells by anti-Thy-1 + C, and direct mixing experiments--we show that T cells are required for the continued suppression of the B cell response to the T-independent antigen, TNP-POL. Suppressor cells can also be induced by TNP-teratoma cells, which lack detectable H-2 antigens. Both anti-Ly-1 + C and anti-Ly-2 + C treatment reversed suppression induced by TNP-SC. These results demonstrate that normal B cell reactivity is present in the spleens of mice rendered tolerant by haptenated self, but that Ly-1,2,3 or Ly-1 + Ly-2,3 suppressor T cells prevent their responsiveness.


Ecto-5'-nucleotidase activity in T and B lymphocytes from normal subjects and patients with congenital X-linked agammaglobulinemia. Ecto-5'-nucleotidase activity was measured in lymphocyte subpopulations isolated from normal subjects and patients with congenital X-linked agammaglobulinemia. B lymphocytes from normal subjects have at least three times more ecto-5'-nucleotidase activity than T lymphocytes. Patients with X-linked agammaglobulinemia have 56% of normal activity in their T cells, and lack a lymphocyte subpopulation high in nucleotidase activity. High activity of ecto-5'-nucleotidase may be a biochemical marker for mature surface immunoglobulin-bearing B cells.


T cell regulation of polyclonal B cell responsiveness. II. Evidence for a deficit in T cell function in mice with an X-linked B lymphocyte defect. In addition to the x-linked B cell maturation deficit previously reported in CBA/N mice, a functional T cell defect has now been observed. T lymphocyte regulation of the polyclonal PFC response was studied within the context of this x-linked immunodeficiency model. The ability of 1) B cells from (CBA X CBA/CaJ)F1, male mice to respond to nonspecific T cell helper signals and 2) T cells from NCF1 male mice to provide such signals was investigated under in vitro conditions by using bacterial lipopolysaccharide (LPS) as the polyclonal activator. B lymphocytes from both male and female NCF1 mice were receptive to T cell help rendered by NCF1 female T cells. Male T cells. however, were unable to augment polyclonal B cell responses of either NCF1 male or female B cells to LPS. Treatment with ATS + C reduced the polyclonal response of female but not male spleen cells to LPS. This deficit could not be overcome by the use of greater numbers of NCF1 male T cells. The observation that this deficiency in T cell regulation is not due to active suppression suggests that the results may be attributable to an intrinsic T cell defect.


Demonstration in vitro of cytotoxic T cells with apparent specificity toward tumor-specific transplantation antigens on chemically induced tumors. Chemically induced tumors of mice exhibit apparently unique antigenicity upon syngeneic transplantation into appropriately immunized hosts. An in vitro counterpart of this pattern in terms of specificity has not been reported. Data are presented that demonstrate that immune peritoneal exudates contain cells cytotoxic for the specific immunogen tumor but with rare exceptions, not toward other syngeneic methylcholanthrene-induced fibrosarcomas. Only tumors highly immunogenic by transplantation criteria induce cytotoxic PEC regularly; nonimmunogenic tumors consistently fail to do so. The effector cell responsible is eliminated by pretreatment with anti-Thy.1 but not anti-Ig plus complement. In concomitant experiments, PEC populations cytotoxic in vitro also conveyed adoptive protection against the specific tumor in syngeneic hosts. This in vitro assay appears to provide a tool for studying T cell-mediated cytotoxicity toward a set of unique surface antigens present on chemically induced tumors.


Subpopulations of human T lymphocytes. XII. In vitro effect of agents modifying intracellular levels of cyclic nucleotides on T cells with receptors for IgM (T mu), IgG (T gamma), or IgA (T alpha). Purified peripheral blood T lymphocytes were incubated with inducers of cyclic nucleotides and examined for the numbers of T cells with receptors for IgM (T mu), IgG (T gamma), or IgA (T alpha). Isoproterenol and theophylline, agents known to increase cAMP levels at 10(-3) to 10(-6) M concentrations, significantly decreased the number of T mu cells but had no effect on T gamma or T alpha cell numbers. This effect of isoproterenol could be completely blocked by a beta-adrenergic blocking agent, propranolol. Receptors for IgM on T mu cells regenerated after treatment with theophylline when cells were washed and further incubated at 37 degrees C over a period of 12 to 24 hr in the absence of theophylline. Phenylephrine, at 10(-3) to 10(-6) M concentrations, significantly increased the numbers of T mu cells but had no effect on T gamma or T alpha cell numbers. The effect of phenylephrine could be completely blocked with an alpha-adrenergic blocking agent, phentolamine. The significance of the results are discussed.


Imbalances of T cell subpopulations in patients with atopic diseases and effect of specific immunotherapy. Reduced numbers of T cells with Fc receptors for IgG (TG cells) are present in blood samples of patients with respiratory allergic disease, mainly those with severe symptoms. TG cells have been previously shown to be suppressor in the pokeweed mitogen- (PWM) dependent B cell differentiation. T cells with Fc receptor for IgM (TM cells), which help immunoglobulin production, are in a normal range. After specific hyposensitization, resulting in a sharp improvement of clinical symptoms, TG cell subset reached normal values.


Ligand-activated T cell growth factor-induced proliferation: absorption of T cell growth factor by activated T cells. The fate in culture of the T cell growth factor (TCGF), which is required for continued growth of human cultured T cells (CTC) in vitro, was studied. TCGF activity was stable for 7 days at 37 degrees C. However, it was no longer detectable after incubation with actively growing CTC at 37 degrees C for 3 days. This loss of TCGF activity also occurred quite rapidly and was detectable within 1 hr of incubation of 0.3 ml supernatant with 2 to 5 x 10(7) CTC at 23 degrees C. 2 x 10(8) mononuclear peripheral blood leukocytes were not effective in removing TCGF activity, and incubation with similar numbers of cells from B and T cell lines had no effect. Three-day-old concanavalin A and phytohemagglutinin blasts were very reactive with TCGF, so that 10(7) or 2 x 10(7) cells consistently removed TCGF activity. These experiments suggested specific absorption of TCGF by activated T cells, and led us to develop a model of ligand-activated TCGF-induced proliferation of T cells: Ligands induce production of TCGF by T-producer cells and deliver a first signal to the T-responder cells. This causes a receptor for TCGF to appear on T-responder cells. Only then does TCGF deliver the obligatory second signal that is needed to drive the T-responder cells into proliferation.


Regulation of antibody response in different immunoglobulin classes. VI. Selective suppression of IgE response by administration of antigen-conjugated muramylpeptides. The suppressive effect of antigen-conjugated muramylpeptides or 6-O-mycoloyl muramylpeptides selectively on the induction of IgE antibody response was demonstrated. Preadministration of DNP-mycoloyl muramylpeptides completely inhibited the induction of the anti-DNP IgE antibody response with DNP-ovalbumin (DNP-OA). The selective suppression of the IgE response was due to the induction of DNP-specific suppressor T cells by DNP-mycoloyl muramylpeptides, and the suppressor cells were shown to be radiosensitive. Preadministration of OA-conjugated muramylpeptides partially inhibited the primary and secondary induction of an anti-OA IgE antibody response. The suppressor effect was also due to the induction of OA-specific suppressor T cells. Application of allergen-conjugated muramylpeptides as therapeutic agents in human allergic diseases was suggested.


Mechanism of cell-mediated cytotoxicity at the single cell level. II. Evidence for first-order kinetics of T cell-mediated cytolysis and for heterogeneity of lytic rate. The kinetics and rate of T cell-mediated cytolysis was assessed by measuring the times required for lysis of isolated target cells by single cytotoxic lymphocytes. Single target cell lysis was determined microscopically by observing trypan blue uptake as a function of time of incubation of effector-target conjugates in agarose. Lysis of EL-4 target cells by alloimmune peritoneal exudate lymphocytes was initiated without a lag and was essentially complete at 2 hr. Both zero-order and first-order kinetics equations were analyzed for fit to the 0 to 2 hr lysis values. Statistically, the zero-order kinetic function could be rejected (p greater than 0.05), but the first-order kinetics function (p less than 0.01) could not. This strong evidence for first-order kinetics of T cell-mediated cytolysis implies that within each CTL-target cell population, cytolysis occurs exponentially as a random decay process and that one event in the entire process of cytolysis is rate limiting. The first-order equation was then applied to measurements of the rate of cytolysis in many different individual effector-target cell combinations. Significant differences in the lytic rate were apparent when either the effector or target cell were varied, with the rate constants spanning a 5-fold range. The heterogeneity of lytic rates is consistent with the hypothesis that lytic efficiency is a function of both the effector and target cells used.


Evidence that thymocytes require at least two distinct signals to proliferate. Optimal receptivity of thymocytes to the mitogenic stimulus in macrophage culture fluid (MCF) is limited to the time in culture when these cells are spontaneously proliferating. By providing a brief, nonmitogenic lectin pulse, nonproliferating cells can be "activated" and their ability to respond to MCF totally restored. This procedure provides a model system for evaluating thymocyte proliferation and, in particular, the mechanism of action of MCF.


Further characterization of the human inducer T cell subset defined by monoclonal antibody. Evidence is presented that the OKTA+ T cell subset in man, defined by a monoclonal hybridoma antibody, provides help for B lymphocyte differentiation in a PWM driven system. Both B cell proliferation and intracytoplasmic immunoglobulin synthesis are facilitated by OKT4+ and not by OKT4- T cells. Given earlier studies demonstrating that OKT4+ T cells were necessary for generation of T cytotoxic cells and the present study that OKT+ T cells are necessary for the differentiation of B cells, it would appear that the OKT+ population is the major human T helper (inducer) subset.


Lack of T-cell mediated cytotoxicity in M-MSV system depending on H-2 haplotypes. The H-2 restriction phenomenon was evaluated with regard to the immune response to Moloney sarcoma virus (M-MSV)-induced tumours in the mouse. Using an in vitro 51Cr release assay the lytic activity of lymphocytes from M-MSV immune strains of mice, including several H-2 recombinant strains, was determined on leukaemic cell lines originally induced in mice of different strains by neonatal infection with Moloney leukaemia virus. In analogy with other experimental models, it was observed that a compatibility between effector and target cells at the K and/or D regions is generally necessary and sufficient to obtain the cytotoxic effect. However, for the H-2d and H-2b haplotypes, identity at the K or D region respectively was not sufficient for lysis to occur. Attempts to demonstrate that this lack of activity might be associated with the absence of H-2 linked Ir responder genes, were not successful.


Concurrent linear scleroderma and systemic lupus erythematosus: a report of two cases. Two patients with linear scleroderma (en coup de sabre) developed systemic lupus erythematosus (SLE). This association has been well documented in only one previous case. The presence of high titer antibodies to ribonucleoprotein (RNP) initially led to the diagnosis of the mixed connective tissue disease. Development of more serious clinical involvement and antibodies to Sm (case 1) or native deoxyribonucleic acid (nDNA) (case 2) helped establish a diagnosis of SLE. Use of these studies in the differential diagnosis of systemic rheumatic diseases is disucssed briefly. The presence of anti-RNAP antibodies in patients with localized scleroderma may herald a more serious rheumatic disease.