Tumour markers and radiological examinations in the follow-up of patients with oral cancer. In 1986, a new follow-up programme for patients with oral cancer was introduced in our department. The follow-up programme included liver function tests, tumour markers and radiological examination. 63 patients were monitored to the end of 1990. The results showed that serum Carcinoembryonic Antigen (CEA) assays were not sensitive enough to detect early cancer, recurrences or metastases. Neither was there any difference between the preoperative CEA levels of patients with and without recurrence during follow-up. The levels of salivary CEA, were similar to those of healthy individuals. Serum CA 19-9 values were consistently normal. In 1 patient, the first sign of liver metastases was a high 5-nucleotidase level. No recurrences were detected by radiological examination. In conclusion, the importance of frequent and careful clinical observation is emphasized; all 20 recurrences at the primary site and in local lymphnodes were detected by clinical examination. For detection of oral cancer recurrences, several laboratory and radiological examinations seem unnecessary. The cost-benefit of those examinations is significantly low.


Proliferating cell nuclear antigen and S phase fraction in endometrial stromal sarcoma. To investigate the value of immunohistochemical staining for the cell cycle protein proliferating cell nuclear antigen (PCNA) and flow cytometric S phase fraction in determining prognosis in endometrial stromal sarcoma, graded according to mitotic count. Seventeen endometrial stromal sarcomas from 13 patients treated at the Royal Marsden Hospital were analysed. Serial 5 microns sections were cut for haematoxylin and eosin and immunohistochemical staining for PCNA, performed using the murine monoclonal antibody PC10. PCNA positivity was expressed as a percentage of the total number of cells (PCNA index). Flow cytometric analysis was performed on nuclei extracted from paraffin wax sections. In the five patients who died of disease within five years, PCNA index varied between < 1% and 60% (mean 21%) and S phase fraction ranged from 11.3 and 20.1 (mean 13.8). Four patients who were apparently cured showed PCNA indices ranging from < 1% to 5% (mean 1.75%) and S phase fraction ranging from 1.4 to 3.5 (mean 2.3); and three patients alive with disease showed PCNA indices ranging from 1% to 15% (mean 8.6%) and S phase fraction ranging from 1.4 to 3.5 (mean 2.3). One patient who died from indolent local disease after nine years showed a PCNA of 1 or less and an S phase fraction of 0.9. PCNA staining was variable and therefore not a reliable prognostic indicator, but a high PCNA index was only found in those patients dying of disease within five years. A stronger association was seen between S phase fraction and prognosis; this also correlated well with histological grade determined by mitotic count. In individual borderline cases that are between low and high grade categories, these procedures may be useful.


HER2 (c-erbB-2) oncoprotein expression in colorectal adenocarcinoma: an immunohistological study using three different antibodies. Paraffin wax sections of 70 surgically resected colorectal adenocarcinomas were examined for the overexpression of HER2/c-erbB-2 oncoprotein using three different specific antibodies and the avidin-biotin immunoperoxidase technique. The patients included 38 men and 32 women aged between 47 and 80 years. The tumours were derived from various parts of the large intestinal tract, and represented all three stages of Dukes' classification and the three histological grades of differentiation. Many tumour sections also included adjacent normal or transitional mucosa. Eight tubular adenomas found in the colectomy specimens in association with some carcinomas were also examined. No positive membrane staining was seen in any of the 70 carcinomas, four adenomas, two hyperplastic polyps, nor in the adjacent normal or transitional mucosa. It is suggested that the overexpression of c-erbB-2 gene product is unlikely to be as common and as pronounced in colorectal adenocarcinoma as it is in ductal carcinoma of the breast.


Glutamate-immunoreactive terminals synapse on primate spinothalamic tract cells. Glutamate has been shown to excite spinothalamic tract (STT) neurons and has been localized to primary afferent neurons, spinal cord projection neurons, and interneurons in the spinal cord dorsal horn. The likelihood that glutamate-immunoreactive (GLU-IR) terminals directly innervate STT neurons was investigated. For these studies three lamina IV or V STT cells in the lumbar spinal cords of three monkeys (Macaca fascicularis) were identified electrophysiologically and characterized. Two were identified as high threshold neurons and one as a wide dynamic range neuron. Following intracellular injection of the cells with HRP and reaction to give the cells a Golgi-like appearance, the tissues were processed for electron microscopy. Postembedding immunogold methods with antibodies specific for glutamate were used to identify GLU-IR terminals apposing the somata and dendrites of the STT neurons, including dendrites that extended into laminae IV and III. The GLU-IR terminals were numerous and constituted a mean of 46% of the population counted that appose the STT soma and 50% of the profiles apposing the dendrites. Fifty-four percent of the somatic and 50% of the dendritic surface length was contacted by GLU-IR terminals. Most terminals contained round clear vesicles and some contained a variable number of large dense core vesicles. For one of the three cells examined it was determined that 45% of the terminals apposing the soma were GLU-IR and 30% of the terminals were gamma aminobutyric acid-immunoreactive (GABA-IR). In an additional monkey, a lamina I cell retrogradely labeled from the ventral posterolateral nucleus of the thalamus was found to be ensheathed in glial processes.(ABSTRACT TRUNCATED AT 250 WORDS)


Sex differences in densities of dopaminergic fibers and GABAergic neurons in the prenatal rat striatum. On the basis of observations on dopaminergic neurons developing in gender-specific cultures of embryonic rat mesencephalon, we have hypothesized that as yet unknown sexual dimorphisms might be found in projection areas of dopaminergic neurons. Therefore we searched for possible sex differences in the striatum during the period when massive ingrowth of mesencephalic afferents occurs and the striatal gamma-aminobutyric acid (GABA)ergic neurons differentiate. Male and female rats of embryonic days (E) 16, 18, 20, and 21 were fixed by perfusion through the heart. Vibratome sections were cut from the striatal anlage and sequentially immunostained for GABA by the immunogold-silver technique and tyrosine hydroxylase (TH) by the avidin-biotin-peroxidase method. Ultrathin sections were scanned for numbers of GABA- and TH-immunoreactive (IR) elements. Densities of TH-IR axons as well as of GABA-IR cell body profiles progressed with time. Contacts between TH-IR axons and GABA-IR and immunonegative cells were observed as early as E-16, increasing in numbers toward later stages. Throughout prenatal development, female striata displayed higher densities of both TH-IR axon and GABA-IR cell body profiles than male ones. This is the first report of a distinct anatomical sex difference regarding two major components of a key center of motor control. Prenatal sexual differentiation of the striatum may lead to a sexually dimorphic extrapyramidal circuitry, the existence of which, in the adult, is suggested by experimental and clinical data.


Expression of cholecystokinin and somatostatin genes in the human thalamus. The cholecystokinin (CCK) gene is expressed in thalamocortical and thalamo-striatal neurons of the rat. In the cat, this peptide is found in some intralaminar and midline nuclei, whereas somatostatin (SRIF) is expressed in the reticular nucleus of the cat but not in rat. Since the putative neurotransmitters used by thalamic neurons are still incompletely known, especially in humans, we investigated the expression of the CCK and SRIF genes in the human thalamus by using hybridization histochemistry. CCK mRNA was found in many neurons, located in several nuclei of the dorsal thalamus. They were especially numerous and widespread in the nuclei associated with the internal thalamic lamina. They formed a continuum in the basal medial thalamus, from the central-medial nucleus, through the centre median/parafascicular complex to the limitans and suprageniculate nuclei. In addition, neurons with CCK mRNA were found medially and laterally to the mediodorsal nucleus, in the midline and intralaminar nuclei. Only rare neurons with CCK mRNA were found in other nuclei (e.g., in the ventral group of nuclei). SRIF mRNA was found in many neurons of the reticular nucleus, but not in the dorsal thalamus. Neurochemical features of the human thalamus, for the genes studied here, resemble those found in the cat. SRIF may play a role in modulating dorsal thalamic impulses, which may be conveyed through CCK innervation to the striatum and, partly, to the cortex.


Molluscum contagiosum in patients with human immunodeficiency virus infection. A review of twenty-seven patients. Molluscum contagiosum (MC) is a common and at times severely disfiguring cutaneous viral infection in patients with human immunodeficiency virus (HIV) infection. The purpose of this study was to describe the clinical course of MC in patients with HIV infection and to examine the relation between presentation of MC and the stage of HIV infection, as measured by T-cell subsets. This is a retrospective case study of 27 patients with MC and HIV infection who had T-cell subset determination within 60 days of diagnosis of MC. The overall mean CD4+ count, CD4+ percentage, and CD4+/CD8+ ratio were 85.7/mm3, 5.9%, and 0.10, respectively. An inverse relation between CD4+ count and the number of MC lesions was observed (p = 0.0023). Fourteen patients (52%) had facial and neck lesions alone, and seven (26%) had lesions in areas associated with sexual transmission. Pneumocystis carinii pneumonia had occurred in 8 patients (31%) and Kaposi's sarcoma in 15 patients (56%). MC can occur as a late manifestation of HIV infection and is a cutaneous correlate of cellular immune deficiency.


Intraepidermal localization of the clone in cutaneous T-cell lymphoma. No immunohistologic techniques are currently available to demonstrate clonality of T-cell lymphomas. Monoclonal antibodies to the variable region of the T-cell receptor (TCR) have been produced that identify minor populations of normal peripheral blood T lymphocytes. We investigated the expression of TCR V-region genes in cutaneous lymphomas to determine whether immunostaining with these antibodies may be a simple method to detect clonal T-cell proliferations and help to distinguish benign lymphoid infiltrates from malignant lymphoma. Cutaneous samples were obtained from 18 cutaneous T-cell lymphomas (14 mycosis fungoides, 1 Sézary syndrome, 2 pleomorphic T-cell lymphoma, and 1 large cell anaplastic lymphoma) and 8 benign lymphoid infiltrates. Frozen sections were incubated with monoclonal antibodies and stained by the alkaline phosphatase-antialkaline phosphatase technique. Staining was performed with a panel of 7 anti-TCR V-region antibodies, 6 T-cell markers, 1 anti-beta chain antibody, and 1 anti-delta chain antibody. Clonality could be demonstrated in 2 of 18 cutaneous lymphomas. We observed the strictly intraepidermal localization of clonal proliferation in one case of early-stage mycosis fungoides. Anti-TCR V-region antibodies may identify a strictly epidermotropic clone in early mycosis fungoides. However, the panel of antibodies currently available stains only a minority of cutaneous T-cell lymphomas. The usefulness of these antibodies as a clonotypic marker needs to be reevaluated when a larger panel of antibodies becomes available.


Cardiovascular and renal effects of eel and rat atrial natriuretic peptide in rainbow trout, Salmo gairdneri. The renal and in vitro vascular effects of atrial natriuretic peptides have been examined in several species of fish. However, comparatively few investigations have described the effects of these peptides on the cardiovascular system in vivo. In the present experiments the dorsal aorta and urinary bladder were cannulated and the effects of atrial natriuretic peptides from rat and eel were monitored in conscious trout during bolus injection or continuous atrial natriuretic peptide infusion. The results show that the initial pressor effect of atrial natriuretic peptides is independent of environmental salinity adaptation (fresh or seawater) and the chemical form of atrial natriuretic peptide injected, but it is affected by the rate of atrial natriuretic peptide administration. This pressor response, and the accompanying diuresis, are mediated through alpha-adrenergic activation. Continuous infusion of either rat or eel atrial natriuretic peptide produces a steady fall in mean arterial blood pressure, which is temporally preceded by an increase in heart rate and a decrease in pulse pressure. Diuresis induced by atrial natriuretic peptides is only partially sustained during continuous infusion. Propranolol partially blocks the increase induced in heart rate by atrial natriuretic peptides, but does not affect either pulse pressure or mean arterial pressure. Propranolol significantly increases urine flow in saline-infused animals but has no apparent effect on animals subjected to infusions of atrial natriuretic peptides. These results indicate that there are multiple foci for the action of atrial natriuretic peptides in trout and that in many instances the effects of atrial natriuretic peptides are mediated through secondary effector systems.


Cutaneous myelofibrosis.

Cutaneous myelofibrosis. Cutaneous extramedullary hematopoiesis is rare, usually occurring in neonates following intrauterine viral infections, hereditary spherocytosis, or the twin transfusion syndrome. Only 20 cases of cutaneous extramedullary hematopoiesis have been reported in adults, all with myelofibrosis. The cutaneous infiltrates may be atypical and difficult to distinguish from leukemia cutis. We have studied a 65-year-old woman with myelofibrosis and approximately 40 violaceous, firm, non-tender cutaneous nodules measuring 1 to 4 cm in diameter, located on her abdomen near a splenectomy scar. Histologically, the lesions had a dense infiltrate of myeloid cells in all stages of maturation, atypical large cells with multilobate nuclei or multiple nuclei, resembling atypical megakaryocytes, and fibroblasts. Although the patient received erythropoietin therapy prior to the development of the nodules, erythroid progenitors were not seen. Reticulin was increased particularly surrounding the atypical megakaryocytes. The myeloid cells stained for chloroacetate esterase and with the polyclonal antibody MAC 387. Atypical megakaryocytes stained for Factor XIIIa and Factor VIII-related antigen. Dendritic Factor XIIIa positive cells were also increased. The skin lesions remain unchanged grossly one year after their development.


The effects of FMRFamide, 5-hydroxytryptamine and phorbol esters on the heart of the mussel Geukensia demissa. The ventricle of the mussel Geukensia demissa is inhibited by 5-hydroxytryptamine and excited by the molluscan neuropeptide FMRFamide. Supra-threshold doses of amide result in marked positive chronotropy and inotropy within 5-15 s. 5-Hydroxytryptamine at 10(-8) M produces diastolic arrest within 10 s. A 1-min exposure to FMRFamide (5 x 10(-8) M) results in a small increase in the cytoplasmic levels of adenosine 3',5'-cyclic monophosphate; shorter or longer exposures have no effect. The cAMP content of ventricles incubated in 5 x 10(-8) M 5-hydroxytryptamine for 1 min decreases by 2.3 pmol/mg protein; longer or shorter incubations have no effect. Treatment with forskolin results in 3- or 4-fold increases in adenosine 3',5'-cyclic monophosphate, but forskolin has no effect on the mechanical activity of the ventricle. The levels of inositol monophosphate, inositol 1,4-diphosphate, and inositol 1,4,5-triphosphate in tissues exposed to 5-hydroxytryptamine are not different from levels in control tissues. FMRFamide decreases the levels of these phosphoinositides by 50% or more. Lower concentrations of phorbol 12,13-diacetate (10(-8) to 10(-7) M) and phorbol 12-myristate,13-acetate (10(-6) M) cause positive chronotropy in the isolated ventricle; higher concentrations induce systolic arrest. These results suggest that the effects of 5HT on the ventricle are not mediated by adenosine 3',5'-cyclic monophosphate or inositol 1,4,5-triphosphate. The effects of FMRFamide may involve a decrease in inositol 1,4,5-triphosphate. The effects of amide may involve a decrease in inositol 1,4,5-triphosphate. The response of the ventricles to phorbol esters suggest that protein kinase C may be involved in the regulation of cardiac contractility.


Separate and additive stimulation of bovine milk yield by the local and systemic galactopoietic stimuli of frequent milking and growth hormone. Lactating heifers were treated for 4 weeks with recombinant bovine growth hormone (bGH, n = 9) or were untreated (n = 9). In addition, two mammary glands of each heifer were milked four times daily rather than the normal twice daily for the same 4 weeks, and for the following 2 weeks. Over the 4 weeks, milk yield was increased 12.8% by bGH, 14.0% by frequent milking and 28.5% by the combined treatment. The effect of bGH as administered here was slower in onset than that of frequency, but eventually produced a higher peak yield. ANOVA revealed significant effects of each stimulus independently and an additive, but not synergic effect of the combined treatment. The effect of the combined treatment tended to persist beyond the end of treatment; most of this response was related to the milking frequency component rather than the bGH. Mammary differentiation was assessed in biopsies of mammary tissue obtained prior to and at the end of treatment. Mammary enzyme activities (expressed on a per cell basis) indicated minimal differentiative response to either treatment, but synthesis rates for lactose and casein determined in vitro were increased by bGH treatment. Histological examination revealed a stimulatory effect of milking frequency on epithelial cell size. The results indicate that these two galactopoietic stimuli operate through independent mechanisms, and neither stimulus alone is sufficient to maximize milk yield in dairy heifers.


Heterogeneity of surfaces of subgingival bacteria as detected by zeta potential measurements. Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans (A.a.) are Gram-negative bacteria which are implicated in various forms of periodontal disease. The Gram-positive Peptostreptococcus micros may also play an important role. For investigation of the possible adhesion and colonization mechanisms of these organisms, the charge properties of the outermost layers of bacterial cell surfaces were studied through the measurement of zeta potentials at various pH values. Eleven fresh clinical isolates, representing the four species, and one laboratory strain, P. gingivalis W83, were examined. Eleven of the 12 strains displayed heterogeneity with respect to pH-dependent zeta potentials. Within single cultures of each of these strains, two distinct populations of cells were found, one which was more negatively charged than the other. For the Gram-negative strains, the more negatively charged subpopulation was in the majority, while the P. micros strains appeared to be composed mainly of a less-negatively-charged subpopulation. Vesicles prepared from two strains displayed the same pH dependence and heterogeneity of zeta potentials as the parent cells. An A.a. strain which was passaged several times in fluid medium had lost its fimbriae and became homogeneous with respect to charge.


Lichen planus annularis: an immunohistochemical study. Lichen planus annularis is a relatively rare skin manifestation of lichen planus. The mechanisms in the formation of annular lesions are not fully understood. We reported here a 57-year-old female with this disease. The eruption initially occurred as lichen-papules, then enlarged (bean-sized, umbilicated small plaques), and finally developed annular manifestations. We performed immunohistochemical examinations of specimens taken from different types of eruptions. In all specimens, HLA-DR was expressed in the focal keratinocytes adjacent to the dermal HLA-DR positive cell infiltration. Both in the initial papule and in the final annular lesion, expression of ICAM-1 was present only in the keratinocytes above the dermal cell infiltration, similar to HLA-DR. It is of interest that, in the umbilicated small plaques, the peripheral epidermis other than the central site extensively reacted to ICAM-1. LFA-1 expression was most prominent in the mononuclear cells impinging on the dermo-epidermal junction in all specimens. In addition, in the periphery of the umbilicated small plaques, which showed no bandlike dense cell infiltration nor degeneration of basement membrane, TNF-alpha, but not LFA-1, was positive in the infiltrated cells of the upper dermis. These results suggest that expressions of ICAM-1 and TNF-alpha in the peripheral keratinocytes and dermal infiltrated cells are important molecular events in the mechanisms of formation of the annular lesions.


Correlation of bronchial eosinophil and mast cell activation with bronchial hyperresponsiveness in children with asthma. Bronchial hyperresponsiveness in asthma has been associated with increased numbers of eosinophils and mast cells in the bronchial airway. It is unclear if these cells are important in the pathogenesis of hyperresponsiveness, and the role of mast cells has been discounted because they are effectively stabilized by beta-adrenergic drugs. Because the pathogenesis of asthma in children may be different from that in adults, and to find out if cellular activation is associated with bronchial reactivity, we studied 17 children with mild to moderately severe chronic asthma who had been treated with intermittent brochodilator therapy and compared their bronchial responsiveness to histamine with the levels of eosinophil cationic protein and mast cell tryptase in broncholavage fluid. The number of eosinophils in lavage fluid was correlated with histamine responsiveness (r = -0.444, p < 0.05) but not with levels of cationic protein (r = 0.33, p = NS). Bronchial responsiveness to histamine was highly correlated with mast cell tryptase (r = -0.714, p < 0.005), but there was no correlation with eosinophil cationic protein (r = -0.355, p = NS). We conclude that in children with chronic asthma mast cells as well as eosinophils contribute to bronchial hyperresponsiveness. Activated mast cells may play a primary role, possibly by tryptase-induced upregulation of bronchial smooth muscle tone.


Influence of long-term hypoxia on tyrosine hydroxylase in the rat carotid body and adrenal gland. Tyrosine hydroxylase (TH) protein was measured in the carotid body and adrenal gland of rats exposed to normobaric hypoxia (10% O2 in nitrogen) for 3, 7, 14 or 22 days. After 22 days of hypoxia, a gradual increase in TH protein content was observed in the carotid body and in the adrenal gland, reaching 168% and 148% of the normoxic controls, respectively. To determine if the increase in TH protein content in the carotid body could alter catecholamine biosynthesis, in vitro TH activity and catecholamine turnover were measured in rats submitted to hypoxia for 14 days. TH activity was increased by 11.2-fold, while the turnover of dopamine and norepinephrine was increased by 14.8- and 5.4-fold, respectively. The data show that long-term hypoxia exerts a stimulatory influence on TH protein in the carotid body and adrenal gland in addition to an elevation in dopamine and norepinephrine biosynthesis in the carotid body.


Acetylcholine receptors in rat cardiac neurones. Membrane potential changes produced by acetylcholine (ACh), and their underlying mechanisms, were studied in neurones of isolated cardiac ganglia of the rat by means of intracellular microelectrodes. Five components of membrane potential change could be detected in cardiac neurones following 1-5 s micro-application of ACh: (i) fast depolarization resulting from an activation of nonselective cationic conductance; (ii) slow depolarization associated with a decreased membrane conductance, presumably for potassium ions; slow hyperpolarization which consisted of (iii) early and (iv) late parts resulting from an activation of calcium-sensitive potassium current and from inhibition of steady-state inward current, respectively; and (v) delayed slow hyperpolarization associated with an increased conductance, most likely for potassium ions. Components (i), (iii) and (iv) persisted in the presence of atropine and were inhibited by nicotinic antagonists. Thus they were due to activation of nicotinic ACh receptors. However, the sensitivity of component (i) to ganglion-blocking agents appeared to be rather low: IC50s for inhibiting (i) were 226 +/- 34.2 microM, 31.2 +/- 4.31 microM and 15.3 +/- 3.27 microM for hexamethonium, d-tubocurarine, and trimetaphan, respectively. Components (ii) and (v) were abolished by atropine (1 microM) and mimicked by muscarine (component (ii) also persisted in d-tubocurarine), hence they resulted from activation of muscarinic ACh receptors. It is concluded that cardiac neurones are endowed with both nicotinic and muscarinic ACh receptors. Their activation leads to membrane depolarization and discharges followed by hyperpolarization and inhibition of discharges.


Biochemical, autoradiographic and functional studies on a unique glutamate binding site in adrenal gland. L-Glutamate is known to function as a major excitatory neurotransmitter in the mammalian central nervous system, and recent reports suggest the existence of receptors for glutamate in several peripheral tissues. In the present study, the characteristics of the binding of [3H]L-glutamate to sections of bovine adrenal gland were studied, and the localisation of this binding was investigated in adrenal glands from cow, dog, rat and guinea pig. In addition, the effects of glutamate on catecholamine release from the perfused isolated bovine adrenal gland were investigated. Binding of [3H]L-glutamate to slide-mounted sections of bovine adrenal gland was of high affinity (Kd 0.4 microM), rapid, saturable, reversible, stereospecific and to a single population of sites. The pharmacological profile of this binding site appeared to be unique, and did not correspond to any of the central receptor subtypes for glutamate so far identified. In the adrenal gland of the cow, rat and guinea pig, the binding density of [3H]L-glutamate was higher in cortex than medulla, while this pattern was reversed in the canine adrenal gland. Glutamate had no effect on the basal secretion of noradrenaline or adrenaline from the perfused isolated bovine adrenal gland, and neither glutamate nor the glutamate receptor antagonist kynurenate altered the nicotine-stimulated release of these catecholamines. These results suggest the existence of a novel peripheral binding site for glutamate in the adrenal gland. The differential autoradiographic localisation of this binding site in the adrenal glands of the various species studied may reflect different functional properties of glutamate in these species, and suggests possible roles for glutamate in the modulation of adrenal function.


Inhibition of metabolic activity of polymorphonuclear granulocytes by thyroid stimulating antibodies. Both thyrocytes and polymorphonuclear granulocytes (PMNs) are known to have on their surface thyrotropin receptor (TSH-R). Anti-TSH-R antibodies having stimulating and blocking effects on thyroid function have been detected in sera of Graves' patients. Thyroid stimulating anti-TSH-R antibodies (TSAb) are involved in the pathomechanism of thyrotoxicosis. The basic question is in this study whether TSAb exerts effects on metabolic activity of PMNs. Immunoglobulin G (IgGs) of 32 patients with thyrotoxicosis had significant inhibitory effect (p less than 0.001) on chemiluminescence of healthy PMNs compared to controls. An inverse correlation was observed between inhibitory effect of IgGs on function of PMNs and TT4 (r = -0.61) in contrast to anti-TSH-receptors antibodies (r = 0.09). TSAbs of eight hyperthyroid patients were measured by determination of cyclic AMP in suspension and slices of porcine thyroid gland. Function of human PMNs separated from healthy donors was determined by luminol-amplified chemiluminescence. Photon-emission of PMNs induced by adherence was significantly lower than that of controls. None of patients' IgGs contained anti-PMNs antibodies or other toxic compounds. It was found an inverse correlation between chemiluminescence of PMNs and capability of TSAb to increase intracellular cAMP in thyroid slices and the thyroid hormone levels of patients with thyrotoxicosis (p = 0.0005). In conclusion, TSH-R on PMNs did not belong to "mute" receptors and metabolic changes in PMNs induced by TSAb might have pathogenetic as well as methodological implications.


Ontogenetic transition of cardiac myosin heavy chain isoforms in rat ventricle: effects of fetal exposure to beta-adrenergic agonists or antagonists. Cardiac myosin heavy chain (MHC) expression undergoes an ontogenetic transition from beta to alpha MHC isoforms. Although thyroid hormone plays a role in this change, the timing of the events suggests the participation of other factors. Using a new, denaturing SDS-PAGE procedure that cleanly resolves the beta and alpha heavy chains, we have assessed the role of beta-adrenergic stimulation on this transition in fetal and neonatal rat hearts. In control animals at embryonic day 20, less than 15% of the MHC was the alpha-form, and the proportion increased to approximately 35% by postnatal day 1 and to 80% by postnatal day 8. Although catecholamine levels rise abruptly at birth, and cyclic AMP levels increase the expression of alpha-MHC in vitro, neither premature beta-adrenergic stimulation (maternal treatment with terbutaline on embryonic days 17, 18 and 19) nor continuous prenatal blockade of beta-receptors (maternal propranolol infusions from embryonic day 7 onward) influenced the developmental profile. Because beta-receptors in fetal and neonatal heart are functionally linked to adenylate cyclase, and cyclic AMP has been shown to promote the expression of alpha-MHC, the lack of effect of terbutaline or propranolol suggests that activation of adenylate cyclase through fetal cardiac beta-receptors is not sufficient to mediate the switchover without participation of other factors, such as thyroid or steroid hormones, or hypoxia.


Discordant segregation of Na+,K(+)-adenosine triphosphatase alleles and essential hypertension. To determine whether the alpha 2 and or beta 1 isoforms of the Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) are involved in the pathogenesis of essential hypertension. Segregation analysis of polymorphic DNA markers was used to test the involvement of Na+,K(+)-ATPase in essential hypertension. Children with persistent hypertension having one parent with essential hypertension were included in the study. Criteria for persistent hypertension were blood pressure readings with systolic and/or diastolic levels exceeding the 95th percentile based upon age and sex. The diagnosis of hypertension for adults, including parents and older siblings, was confirmed using criteria recommended in the 1988 report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure. In three essential hypertensive families consisting of 18 members including 11 hypertensives, several obligate recombinants between the Na+,K(+)-ATPase alpha 2 isoform marker and the hypertension phenotype were observed. Similarly, in one hypertension family consisting of four members, obligate recombinants between the beta 1 isoform marker and the disease were observed. The discordant segregation of the alpha 2 and beta 1 isoform markers and essential hypertension suggests that neither the Na+,K(+)-ATPase alpha 2 nor beta 1 isoform genes play a primary role in the pathogenesis of hypertension in the families studied.


Review of the management of open fractures. This article presents a literature review of the evaluation and management of open fractures. A case report of a patient having sustained a type II open fracture of the hallux is presented. Debridement, tetanus prophylaxis, reduction and stabilization, and intravenous antibiosis are the hallmarks for prompt and appropriate treatment.


The frequency of alpha 2-adrenoceptor restriction fragment length polymorphisms in normotensive and hypertensive humans. To examine the frequency of distribution of allelic polymorphisms of the alpha 2-adrenoceptor gene in normotensive and hypertensive humans. The frequency of alpha 2-adrenoceptor genotypes was compared in the two groups using the chi 2-test. The Midwest Hypertension Research Center Outpatient Clinic of Creighton University School of Medicine. History was taken from and physical examination performed on each of the 60 hypertensive and 47 normotensive adults. DNA was extracted from leukocytes from each participants. Twenty restriction endonucleases were used and one restriction fragment length polymorphism (RFLP) was found using a 950-bp restriction fragment from the coding region of the human platelet alpha 2-adrenoceptor gene (ADRA2R) and Bsu36I restriction endonuclease. This probe and Bsu36I restriction endonuclease, in addition to another restriction endonuclease (Dra I), were then used in the study. Three genotype patterns were found. Homozygotes for the Bsu36I RFLP have either a unique 12-kb or a unique 5.8-kb band. Heterozygotes have both bands. The frequency of this alpha 2-adrenoceptor RFLP was calculated. In hypertensives the frequencies of the 12- and 5.8-kb alleles were 0.52 and 0.48, compared with 0.45 and 0.55, respectively, in normotensive, a difference that was not statistically significant. The frequency of the Bsu36I RFLP involving an alpha 2-adrenoceptor gene in hypertensives did not differ significantly from that in normotensives. A genetic linkage study is now under way to test for an association of the Bsu36I RFLP of the alpha 2-adrenoceptor gene with essential hypertension in families.


The potentiation of narcotic analgesics with phenothiazines. Potentiation of narcotic analgesics with phenothiazines have been used for preoperative and postoperative analgesia for a number of years by the podiatric community. The agents most frequently cited in studies are the combination of meperidine with either the phenothiazines chlorpromazine, trimeprazine, or promethazine. Due to the fact that the double-blind clinical studies of pain are mainly subjective, and difficult to quantify, the usefulness of phenothiazines for the sole purpose of potentiation of analgesia is questionable, especially in view of the many side effects of this group of drugs.


Changes in the precursor frequencies of IL-4 and IFN-gamma secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis. Limiting dilution analysis was used to estimate the frequency of clonogenic Ag-specific CD4+ T lymphocytes in draining lymph nodes of mice over the course of infection with Leishmania major, and to measure the production of IL-2, IL-3, IL-4, IFN-gamma, and TNF by the resultant clones. Infection of both genetically susceptible BALB/c ("non-healer") and resistant C57BL/6 ("healer") mice resulted in at least a fourfold increase in the frequency (to about 0.3%) and at least a 10-fold increase in the total number of lymph node CD4+ cells that formed clones when cultured with L. major Ag in vitro. At 1 wk after infection, the majority of clones from BALB/c mice secreted IL-4 (precursor frequency 0.15%) and fewer secreted IFN-gamma (0.05%); this pattern remained constant for at least 8 wk after infection. In C57BL/6 mice, however, a high precursor frequency of IL-4-secreting clones was measured in the first 1 to 2 wk when the mice had lesions, but resolution of infection was associated with a decrease in the frequency of IL-4-secreting clones (from 0.13% at 2 wk to 0.03% at 4 wk) and an increase in the frequency of IFN-gamma-secreting clones (from 0.08% to 0.22%). At all stages of infection, most clones from either mouse strain secreted IL-3 and very few secreted TNF. Analysis of PCR-amplified cDNA from draining lymph nodes of infected mice also revealed that IL-4 and IFN-gamma mRNA were expressed in both mouse strains early in infection. IL-4 mRNA was the major species at 2 and 6 wk after infection in BALB/c mice, but declined relative to IFN-gamma mRNA over this time in C57BL/6 lymph nodes. Precursor frequency estimates of lymphokine-secreting CD4+ cells in draining lymph nodes therefore correlated with lymphokine expression patterns in vivo. Analysis of a panel of individual short term clones derived from mice 1 wk after infection revealed marked heterogeneity in lymphokine production patterns. In BALB/c mice, 49% secreted IL-4 without IFN-gamma, 18% secreted IFN-gamma without IL-4, and 14% secreted both IL-4 and IFN-gamma. Similarly in C57BL/6 mice, 39% secreted IL-4, 20% secreted IFN-gamma, and 17% secreted both lymphokines. Many of the clones also produced IL-3 and/or IL-2. Together the data suggest that both IL-4 and IFN-gamma are synthesized early in infection of susceptible and resistant mice as assessed by mRNA and precursor frequency analyses.(ABSTRACT TRUNCATED AT 400 WORDS)


Anergic Th1 cells express altered levels of the protein tyrosine kinases p56lck and p59fyn. Tolerance in T lymphocytes can result from clonal anergy, or paralysis, of Ag-specific T cells. To investigate the molecular mechanisms responsible for anergy, a system in which tolerance can be induced in vitro was employed. Anergy, as defined by long-lived nonresponsiveness to normal antigenic stimulation for IL-2 production, was produced in cloned murine CD4+ Th1 cells. Here we report that such anergic Th1 cells express constitutively reduced amounts of the protein tyrosine kinase p56lck and constitutively elevated levels of the protein tyrosine kinase p59fyn. Because protein tyrosine phosphorylation is known to be important for the normal induction of IL-2 synthesis, these results suggest that T cell anergy may be maintained, at least in part, by alterations in tyrosine phosphorylation signaling events.


Restriction fragment length polymorphism of the murine C4 and Slp genes: two C4 groups. We have examined the related H-2 genes coding for the fourth component of complement (C4) and the sex-limited protein (Slp) from 30 inbred mouse strains by Southern blot analysis. With four restriction enzymes, 11 RFLP patterns distributed among 26 different H-2 haplotypes have been identified. Strains of the same serologic H-2 haplotype were found to have identical RFLP patterns. It was confirmed that the number of C4-related genes in most haplotypes is two, Slp and C4; but H-2SWI6 (SWI6) and SWI9, which have the same RFLP pattern, have four and Sw7 has five. Although C4 and Slp have many similarities, they also were found to contain distinctive features: relative to Slp, each C4 allele examined has two insertions totaling 1.1 kb located in introns 14 and 15; and each Slp allele examined, excluding hybrids, has a provirus insertion upstream. No other large deletions or insertions were detected. The RFLP patterns are also due to 10 polymorphic restriction sites, which have been placed on standard maps; two are associated with Slp and eight are associated with C4.Sk strains, the only strains that express low serum levels of C4, have the same RFLP phenotype as Sw14, Sw18, and Swx; Sk may have arisen from a recent common ancestor of these strains. Homologous recombination has been important in the formation of existing C4 alleles. However, based on complete linkage disequilibrium between three RFLP internal to C4, the haplotypes have been divided into two groups that may have functional significance.


High levels of IL-2 alter signal transduction in cloned IL-4-producing CD4 T cells. Cloned CD4 T cells of the Th2 type make IL-4 and related cytokines upon receptor cross-linking, whereas cloned CD4 T cells of the Th1 type make IL-2, IFN-gamma, and TNF-beta. These two types of CD4 T cell are also reported to use distinct mechanisms of signal transduction. It has been reported that Th1 cells flux Ca2+ upon receptor cross-linking, whereas Th2 cells do not. We have noted that when cloned Th2 cells are exposed to high levels (20 U/ml) of IL-2, they show an altered phenotype. Such cells are much more sensitive to activation by certain antireceptor antibodies, they flux calcium upon receptor ligation without additional cross-linking with anti-Ig antibodies, and they make much larger amounts of IL-4. In addition, the organization of their TCR is altered, with increased levels of the TCR-eta chain and an increase in the extent of association of CD4 with CD3 and CD45, changes similar to those found in Th1 cells. These results suggest that there is no fundamental difference in the signal transduction apparatus of Th1 and Th2 cells; rather, the IL-2 made by Th1 cells may create similar phenotypic changes in these cells and thus create the impression of altered signal transduction mechanisms. These results do show that exposure to high levels of IL-2 can profoundly affect signal transduction in T cells. Furthermore, we found that the Ca2+ signal caused by CD3 antibodies seemed to differ in character from that caused by TCR antibodies suggesting that the use of CD3 antibodies is not always a good model for activation through the TCR.


Depletion of CD4+ T cells but not inhibition of the protective activity of IFN-gamma prevents cure of toxoplasmosis mediated by drug therapy in mice. Toxoplasmosis is a frequent opportunistic infection in patients with AIDS. In these patients the major immune deficiencies are a severe depletion of CD4+ T lymphocytes and an impaired capacity to produce IFN-gamma. A mouse model was developed and used to study the effects that depletion of CD4+ T cells and/or inhibition of the protective activity of IFN-gamma have on the effectiveness of the drug therapy for toxoplasmosis. Infection of mice with a lethal inoculum of Toxoplasma gondii cysts followed by treatment with the hydroxynaphthoquinone 566C80 or with sulfadiazine resulted in 100% survival whereas untreated controls had 100% mortality within 15 days of infection. Administration of antiserum to IFN-gamma resulted in early death of untreated mice and in 30% mortality in those treated. Administration of mAb to CD4+ T cells followed by infection with T. gondii prevented the development of both antibody and cell-mediated immune responses against the parasite. These mice resisted the acute infection while undergoing specific treatment. Discontinuation of the treatment, however, resulted in reactivation of the infection and the majority of the animals died within 17 days of suspension of the treatment. Administration of antiserum to IFN-gamma or to CD4+ T cells 24 h but not 15 days after conclusion of the treatment also resulted in mortality. These results indicate that successful treatment of toxoplasmosis depends on the status of the immune system, particularly of CD4+ T cells. Although it is speculative to compare results obtained in mice to the situation in humans, our work suggests that restoration of a competent immune response is of crucial importance for a successful treatment of toxoplasmosis in immunocompromised individuals.


Predominance of Th1 cells in ocular tissues during herpetic stromal keratitis. Herpetic stromal keratitis (HSK) appears to represent an immunopathologic response in the cornea of the eye to HSV-1. T cells of the CD4+ subset were shown to be involved in the mediation of HSK, but how they subserve an immunopathologic role is uncertain. In the present report, we have isolated cells from eyes in the active phase of HSK and studied their cytokine profile after culture in vitro or stimulation with Ag or nonspecific mitogens. Inflammatory cells recovered from eyes consist of polymorphonuclear leukocytes, macrophages, and lymphocytes. As reported before, all the lymphocyte recovered were of the CD4+ phenotype. After stimulation in vitro with Ag or mitogen the cytokines IL-2, IFN-gamma, and TNF-alpha/beta were produced, but not the cytokines IL-4 and IL-10. Thus, on the basis of cytokine profile, ocular lymphocytes were identified as Th1 cells. Ocular cells were also stimulated with PMA and shown to produce IL-1. The results were discussed in terms of the possible means by which the Th1 cells induce tissue damage in HSK as well as in terms of the possible means by which a preferential accumulation of Th1 cell occurs in the eye.


Selective decrease of CD26 expression in T cells from HIV-1-infected individuals. The decrease of CD4+ cells in AIDS patients is widely documented, although the selective loss within different subsets of CD4+ cells and the mechanisms involved in this phenomenon are controversial. In the present report we have analyzed the proliferative response to Ag and mitogen of peripheral blood T lymphocytes from HIV-infected individuals, the phenotype profile of CD26+ and CD26- subset of cells and their infectivity by the HIV. The expression of CD26 Ag, either in CD4+ or CD8+ cells, was clearly diminished in all the patients tested. On the other hand, the expression of CD29 seems not to be affected, nevertheless T cells from these patients were unable to generate a proliferative response against soluble Ag. In 11 out of 13 patients, polymerase chain reaction studies demonstrated that the CD26- subset of CD4+ cells was the main reservoir for HIV-1 in infected individuals and HIV-1 virus preferentially infected in vitro CD4+/CD26- subpopulation. This capacity for preferential infectivity, together with the selective loss of cells expressing CD26 Ag, helps to explain the progressive impairment in the immune system of these patients and sheds new light on our understanding of the AIDS pathophysiology.


Development of murine lupus in CD4-depleted NZB/NZW mice. Sustained inhibition of residual CD4+ T cells is required to suppress autoimmunity. Chronic administration of anti-CD4 mAb prevents autoimmune disease in NZB/NZW F1 (B/W) mice. This may be due either to CD4 cell depletion or to inhibition of CD4 cell function. To evaluate the relative importance of these mechanisms, we devised a system in which the consequences of cell depletion could be analyzed independent of the inhibitory effects of chronic mAb therapy. This was accomplished by performing adult thymectomy before mAb administration. Specifically, female B/W mice underwent thymectomy or sham thymectomy at age 6 wk, followed at age 3 mo by a short course of either anti-CD4 (2 mg/wk for 3 wk) or saline. Treatment with anti-CD4 depleted 90% of circulating CD4 cells, but a small subpopulation (10%) of CD4 cells was refractory to depletion. In non-thymectomized mice, the CD4 population gradually reconstituted after cessation of therapy. In contrast, in thymectomized mice, recovery of CD4 cells was prevented by the absence of the thymus. Despite the striking reduction in CD4 cells in thymectomized mice, severe autoimmune disease developed, with autoantibody levels, proteinuria, and mortality comparable with non-thymectomized, nondepleted controls. The unexpected development of lupus nephritis in thymectomized, CD4-depleted B/W mice suggested that the thymus might be required to achieve the benefits of therapy with anti-CD4. To exclude this possibility, we demonstrated that chronic therapy with anti-CD4 prevents autoimmunity in thymectomized B/W mice. These findings imply that: 1) substantial depletion of CD4 T cells is not sufficient to suppress autoimmunity; 2) suppression of autoimmunity requires sustained functional inhibition of CD4 T cells; and 3) a small subpopulation of CD4 cells that is refractory to depletion by anti-CD4 is sufficient to promote the full expression of murine lupus in B/W mice.


Consistency of routine measurements of CD4+, CD8+ peripheral blood lymphocytes. In order to evaluate the reliability of CD4 and CD8 T lymphocyte counts in large scale studies, a quality control study was performed in 12 French laboratories. CD4 and CD8 counts, assessed by various haematological and immunological techniques, were compared in order to assess possible differences between the laboratories and the techniques used. Our data showed that (a) the consistency of CD4 measurements was satisfactory since the between-laboratory coefficient of variation for absolute CD4 cell numbers above 200/mm3 was around 15% instead of 5-10% for all laboratories but one; (b) the major sources of variability arose from the use of automatic devices in the two-step measurement procedure: immunophenotyping and haematological counting. These data suggest that multicentre assays of CD4 and CD8 counts result in some increase in their variability. Nevertheless the results of large multicentric trials can be extrapolated with confidence in the routine care of HIV+ patients. Together, the results justified the involvement of several experienced laboratories in a clinical trial of HIV-related disease.


Interaction of Citrobacter diversus strains with HEp-2 epithelial and human umbilical vein endothelial cells. More than 75% of neonates with Citrobacter diversus meningitis develop brain abscesses. Interaction of C. diversus strains with HEp-2 and human umbilical vein endothelial cells (HUVEC) was studied to examine mechanisms related to brain abscess formation. Two of 9 strains invaded HEp-2 cells and 0 of 6 invaded HUVEC better than the others. C. diversus survived at least 20 h within HEp-2 cells (in decreasing numbers). Adhesion to HEp-2 cells was increased in 3 of 4 strains expressing type 1 fimbriae, but this did not correlate with increased invasion. Inhibition of RNA or protein synthesis blocked invasion but not adhesion. Thus, invasion requires ongoing protein synthesis, and adhesion to and invasion of HEp-2 cells by type-1-fimbriated strains are independent steps. Invasion was inhibited by cytochalasin D. A 32-kDa protein found in cerebrospinal fluid isolates of C. diversus was not related to invasion of either cell line. Ability to invade HEp-2 cells was not increased among strains isolated from central nervous system sources.


Escherichia coli in fecal flora of healthy adults: serotypes, P and type 1C fimbriae, non-P mannose-resistant adhesins, and hemolytic activity. Escherichia coli strains from feces of 287 healthy adults were analyzed for O:K serotypes, P and type 1C fimbriae, non-P mannose-resistant adhesins, and hemolysin production. O1, O2, O6, O7, O18, O25, and rough-type lipopolysaccharide accounted for 40% of all isolates. K antigen was present in 62%; K1 was identified in 30% and K5 in 10%. O1, O2, O18, rough lipopolysaccharide, and P fimbriation were significantly associated with K1 and O6, O25, and type 1C fimbriation with K5. The overall prevalence of virulence-associated determinants was 22%; 7% of the strains possessed two or three of them. These determinants were most often associated with 9 serotypes (O1:K1, O2:K1, O6:K1, O6:K5, O7:K1, O16:K1, O22:K-, O25:K5, and R:K1) among which their prevalence was 43%, three times greater than among the remaining strains (P < .001). No enteropathogenic or enterohemorrhagic E. coli-associated serotypes were detected.


Early signs of carotid and iliac atherosclerosis in patients with severe hyperlipoproteinemia. Hyperlipidemia is a major risk factor for atherosclerosis. Early signs of cardiovascular disease can be detected also in asymptomatic patients with hyperlipidemia. Forty-four patients with serum cholesterol greater than 300 mg/dl (7.8 mmol/l) and/or serum triglycerides greater than 500 mg/dl (5.6 mmol/l) and 35 healthy controls had their carotid and iliac arteries examined by echo-Doppler with spectral analysis. Systolic ankle pressure was also measured. A vascular score was calculated: a 0 score was assigned to normal findings and a 1 score for each artery with abnormality at echo-Doppler or Winsor index less than 0.97. The XbaI Restriction Fragment Length Polymorphism of Apo B gene was investigated in all hyperlipidemic patients. Arterial lesions, especially those of internal carotid and iliac arteries, were more frequent (p less than 0.01) in patients with high serum lipids, compared to controls. Patients with lesions were older and had higher serum triglyceride concentrations compared to those without lesions. When divided according to serum triglycerides, patients with concentrations exceeding 200 mg/dl had higher vascular score (p less than 0.02) compared to those with serum triglycerides less than 200 mg/dl. No difference in restriction fragment length polymorphism (XbaI) of Apo B gene was demonstrated in patients with arterial lesions compared to those without lesions. Non-invasive echo-Doppler examination gives useful information on the arterial involvement of hyperlipidemic patients and its use should therefore be implemented, especially when high triglyceride concentrations are present.


The filter does not act as a DNA size discriminator in the neutral filter elution technique. Various types of filters were used with the neutral filter elution technique. No significant differences in elution rate or curve shapes were found. Therefore we assume that the release of DNA fragments from the DNA gel formed by lysis of cells is the rate-determining event in elution and not the rate of migration through the filter. We suggest that the filter only serves as a support for the DNA gel.


Effects of trypanothione on the biological activity of irradiated transforming DNA. Held et al. (1984a,b) demonstrated previously that glutathione (GSH), a negatively charged thiol, is significantly less efficient in the hydrogen atom donation repair reaction with radicals induced by radiation in transforming DNA (t-DNA) than are other thiol compounds. Fahey et al. (1991a,b) postulated that the charge on thiols can influence their ability to radioprotect DNA. GSH, which is excluded from the vicinity of DNA due to its negative charge, is less protective than neutral or positively charged thiols. We have investigated this phenomenon further with trypanothione, the conjugate of glutathione and spermidine, N1,N8-bis (L-gamma-glutamyl-L-hemicystinyl-glycyl)-spermidine. Trypanothione exists in aerobic solution largely as the disulphide (T(S)2) but is maintained in the cell in the reduced form (T(SH)2) by means of an NADPH-dependent flavo-enzyme, trypanothione reductase (TR). Experimental data show that T(S)2 in the presence of TR radioprotects t-DNA in the absence of oxygen much better than GSH or spermidine alone or in combination. Little radioprotection by T(S)2 is seen when TR is not present. The results obtained with reduced trypanothione at low concentrations suggest that radioprotection of t-DNA in hypoxia occurs predominantly by H atom donation and slightly by .OH radical scavenging, and the protection is greater than that by GSH or spermidine because the polyamine moiety in trypanothione allows a greater concentration of GSH near the DNA molecule.


Effect of 2-deoxy-D-glucose on DNA double strand break repair, cell survival and energy metabolism in euoxic Ehrlich ascites tumour cells. Effects of 2-deoxy-D-glucose (2-DG) on DNA double strand break (dsb) repair, cell survival and on the energy metabolism were investigated in exponentially growing Ehrlich ascites tumour (EAT) cells. Cells in suspension were exposed to 40 Gy of X-rays and allowed to repair (up to 4 h) with or without 2-DG at 37 degrees C. DNA dsb rejoining was measured by means of clamped homogeneous electric field (CHEF), a pulsed field gel electrophoresis technique. The fraction of activity released (FAR) during electrophoresis (DNA associated 14C-thymidine) was used as a parameter to determine the number of dsb present in the DNA. Biphasic kinetics for dsb repair were observed. The presence of 2-DG significantly inhibited the slow component of dsb repair. The presence of 2-DG also enhanced radiation-induced cell killing. ATP content of cells was measured by a bioluminescence method. ATP content in exponentially growing cells was about 4 pg per cell. The level of ATP was reduced by 50% in presence of 2-DG (C2-DG/CG = 1.0).


Synergistic effect of aphidicolin and 1-beta-D-arabinofuranosylcytosine on the repair of gamma-ray-induced DNA damage in normal human fibroblasts. The effects on enzymatic DNA repair of aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), two potent inhibitors of long-patch excision repair, were investigated in cultured human cells exposed to 60Co gamma-radiation. Using alkaline-sucrose velocity sedimentation analysis, both drugs were shown to inhibit markedly the repair of radioproducts in cultures exposed to greater than or equal to 150 Gy, indicating that a significant component of gamma-ray-induced DNA damage is operated on by a long-patch excision pathway. Moreover, while the extent of repair inhibited by aphidicolin was comparable to that suppressed by araC, combined exposure of irradiated cultures to the two drugs elicited a synergistic response. Specifically, in all three normal fibroblast strains examined, the yield of aphidicolin- or araC-detectable sites (lesions whose repair could be blocked by each drug alone) observed during the first 2 h after irradiation with 150 Gy ranged from 0.8 to 1.2 per 10(8) daltons genomic DNA, whereas the incidence of sites detected by combined exposure to the inhibitors was increased 4-fold (i.e. 3.8 per 10(8) daltons). This difference in site yield leads us to propose that simultaneous administration of aphidicolin and araC serves to block, in addition to long-patch repair, a second mode of excision repair which is refractory to each drug alone.


Enhanced excision repair activity in mammalian cells after ionizing radiation. Monkey CV-1 cells which had received 5 Gy 12 h before harvesting lysates from their cell cultures contained approximately three times as much DNA excision repair enzyme activity as unirradiated cells. The activity was determined in crude cell lysates by the release of intermediate mobility DNA fragments and fragments with 3'-phosphoryl ends from 5'-32P-end labelled irradiated 95 bp alpha DNA. Different 3'-termini endow the fragments with differing mobilities, signifying steps in the processing of radiation damaged DNA. Similar results were obtained when Krebs II mouse tumour cells growing in mice as ascites received 5 Gy 12 h before harvest. The enzyme activities from CV-1 cells and from Krebs II cells were partially purified as 60-70 kDa proteins on Superose 12 or Ultrogel AcA-54 columns. Divalent cations were not required for enzyme activity. A 23 nucleotide long defined duplex oligodeoxynucleotide substrate containing a single 8-oxodG residue was also very actively cleaved by the partially purified cell enzymes. 8-oxoguanine is a major product of ionizing radiation's action on DNA and was recognized by the enzymes described here. The mechanism by which radiation increased excision repair activity of cellular enzymes is not understood.


Hepatocyte response to continuous low dose-rate radiation in radioimmunotherapy assessed by micronucleus assay. The response of hepatocytes to low dose-rate irradiation was examined in mice following the injection of radiolabelled monoclonal antibody. Mice were injected intravenously with an 131I-labelled monoclonal antibody 196-14 which recognizes CA125 antigen, and the effect of continuous low dose-rate irradiation on hepatocytes was assessed using the micronucleus assay. The frequency of micronuclei increased in a dose-dependent fashion, but it was lower than the frequency induced by conventional external X-rays which was determined immediately after the irradiation. A linear quadratic model (micronucleus frequency = aD+bD2+c) showed that the value of b decreased with low dose-rate irradiation from the radiolabelled antibody. It is concluded that the micronucleus assay is useful for the evaluation of the response of hepatocytes to irradiation in radioimmunotherapy.


Modelling the formation of polycentric chromosome aberrations. Exchange-type chromosome aberrations produced by ionizing radiation or restriction enzymes are believed to result from pairwise interaction of DNA double-strand breaks (dsb). In addition to dicentrics, such aberrations may include higher-order polycentrics (tricentrics, tetracentrics, etc.). We have developed computer programs that calculate the probability of the various polycentrics for a given average number of pairwise interactions. Two models are used. Model I incorporates kinetic competition between restitution, complete exchanges (illegitimate recombination events), and incomplete exchanges. Model II allows unrestituted breaks even if there is no recombination. The models were applied to experimental observations of aberrations produced in G1 Chinese hamster ovary cells after electroporation with the restriction enzyme PvuII, which produces blunt-end dsb. We found, experimentally and theoretically, that there was a maximum in the number and multiplicity of polycentrics per cell: beyond a certain PvuII concentration no additional or higher-order polycentrics were produced. Computer-generated relationships, which were remarkably similar for both models and for all values of the adjustable parameters, were found between dicentrics per cell and higher-order polycentrics per cell. Excellent agreement was found between the experimental observations and the consensus theoretical curve relating tricentrics per cell to dicentrics per cell. The observed number of higher polycentrics per cell for a given number of dicentrics per cell was somewhat larger than the consensus theoretical prediction. The observed number of centric rings per cell was markedly larger than the consensus theoretical value, presumably owing to intrachromosomal localization ('proximity effects'). The computer models also provided estimates for the adjustable parameters; for example, in model I the fraction of incomplete exchanges was found to be about 35%.


Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains. A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40 T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D0 = 192 +/- 13 vs 127 +/- 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. A retinoblastoma susceptibility gene (RB) binding defective mutant showed moderately increased radioresistance (D0 = 174 +/- 10). However, cell clones transfected with three different p53 binding defective mutants showed no change in radiosensitivity (D0 = 132 +/- 5) as compared with neo gene transfected controls. Transfection with T-antigen mutants defective in either the RB or p53 binding domain yielded no morphological alterations characteristic of transformation. These data suggest that the SV40 T/p53 complex may be of importance in the radioresistance phenotype.


Intrinsic radiosensitivity and PLD repair in osteosarcoma cell lines. The response to radiation of seven osteosarcoma cell lines was analysed by in vitro colony-forming assay and compared with that of eight human fibroblast strains. The values of D0, the surviving fraction after 2 Gy (S2Gy), and the mean inactivation dose (D) of osteosarcoma cells in log-phase culture were significantly higher than those of fibroblast strains (p less than 0.01). PLD (potentially lethal damage) repair of osteosarcoma cells evaluated in the plateau phase of growth showed great variation for enhancement of survival, although all of the values were maximized within 12 h after irradiation. In the osteosarcoma, intrinsic radiosensitivity in vitro reflected the clinical response to radiation. However, the capacity for PLD repair might not be a good indicator for predicting the results of radiation therapy.


Radiation-induced apoptosis of oligodendrocytes in vitro. It has been suggested that glial cells and/or their progenitors are the primary target cells for radiation-induced demyelination. Cultures of terminally differentiated oligodendrocytes, immature oligodendrocytes, and O-2A progenitor cells were generated from the cerebral cortex and spinal cord of perinatal rat pups. Irradiation of cultures of terminally differentiated oligodendrocytes resulted in a significant increase in the percentage of apoptotic cells from 15% in control to 30% in irradiated samples, with the maximum increase induced by 10 Gy. This increase in apoptosis could be observed by 1 h after irradiation with the maximum level reached at 3-6 h. Apoptotic cells were not detected before or after irradiation of cultures of O-2A progenitor cells or immature oligodendrocytes. These data suggest that radiation-induced apoptosis of terminally differentiated oligodendrocytes may be involved in early demyelination.


Ascorbate-assisted, phthalocyanine-sensitized photohaemolysis of human erythrocytes. The rate of photohaemolysis of human red blood cells sensitized by chloroaluminium phthalocyanine sulphonate is increased by ascorbate, with or without added FeCl3. Stimulation of haemolysis by ascorbate without addition of metal salt, and in the presence of a strong chelator such as desferrioxamine, is an unexpected phenomenon. Lysis rate and ascorbate concentration were directly related, suggesting that ascorbate acts as a reactant and not as a catalyst. The process also requires oxygen; azide and D2O tests indicate some participation of singlet oxygen, although to a lesser extent than in the photosensitized haemolysis in the absence of ascorbate. Kinetic considerations suggest a reaction path initiated by excited sensitizer and ascorbate, parallel to the singlet oxygen-mediated process. Because of the ubiquitous presence of ascorbate in human tissues in concentrations comparable to those of dissolved oxygen, it is quite possible that in photodynamic therapy a fraction of the photodynamic damage proceeds via a Type I, ascorbate-assisted, mechanism.


The efficacies of 3,4,3-LIHOPO and DTPA for enhancing the excretion of plutonium and americium from the rat: comparison with other siderophore analogues. With DTPA as a comparison, the siderophore analogue code named 3,4,3-LIHOPO has been tested for its ability to remove 238Pu and 241Am from rats after their inhalation or intravenous injection as nitrate. The most effective treatment regimen for inhaled Pu was the repeated administration of 30 mumol kg-1 3,4,3-LIHOPO. By 7 days after exposure, the Pu contents of the lungs and total body were reduced respectively to 2 and 4% of those in untreated animals. These values were six and three times less than when DTPA was administered using the same protocol. For inhaled Am, 3,4,3-LIHOPO and DTPA were considered equally effective, the lung and total body contents being reduced respectively to 13 and 10% of those in controls. Some animals showed slight degenerative changes in the liver and proximal tubules of the kidneys after the repeated administration of 30 mumol kg-1 of 3,4,3-LIHOPO; however these changes were less marked than after DTPA treatment. After the intravenous injection of Pu, the most effective regimen was the single administration of 3 mumol kg-1 3,4,3-LIHOPO. The body content at 7 days was reduced to 7% controls compared with 19% after the repeated administration of 30 mumol kg-1 DTPA. At a dosage of 30 mumol kg-1, 3,4,3-LIHOPO was less effective owing to the higher retention of Pu in the liver. With repeated dosages of 30 mumol kg-1 3,4,3-LIHOPO was more effective than DTPA for the decorporation of Am; the body contents were 16 and 31% of those in controls respectively. Importantly, the body content was still reduced to 28% of control after a single administration of 3 mumol kg-1. The ligand 3,4,3-LIHOPO, which is also superior to other siderophore analogues, could represent a most significant development in the decorporation of Pu and Am.


Effects of lofexidine, an alpha 2-adrenoreceptor agonist, on ocular blood flow and ion transport of rabbit iris-ciliary body. The effect of topical unilateral application of lofexidine, an alpha 2-agonist, on ocular regional blood flow was tested in anesthetized rabbits using radiolabeled microspheres. A significant reduction in blood flow was found only in the ciliary body of the treated eye at 1 hr after lofexidine treatment. However, the IOP of both eyes was decreased significantly at 30 and 60 min post lofexidine treatment. Yohimbine (i.v.) blocked this IOP lowering effect, but only partially prevented the blood flow response. In addition, no significant difference was found in either basal or amphotericin-B stimulated short-circuit current or potential difference of the isolated rabbit iris-ciliary body after 30 minutes of lofexidine treatment. These observations suggest that the IOP lowering effect of lofexidine appears to be mediated by alpha 2-receptors but unrelated to the reduction in the ocular blood flow and net electrogenic ion transport.


Studies on the mechanism of immunologic tolerance induction by murine dendritic epidermal Thy-1+ cell lines. We are investigating the functions of the Thy-1+ dendritic cells present in murine epidermis. Dendritic epidermal Thy-1+ cell (DETC) lines conjugated in vitro with hapten induce specific immunologic tolerance upon intravenous (i.v.) or subcutaneous injection into the footpad of normal mice. In these studies, we demonstrate that hapten-conjugated cells of other long-term, interleukin-2 (IL-2)-dependent, T cell lines are unable to induce tolerance upon footpad injection, indicating that the ability of DETC lines to induce tolerance is not a function of long-term cell culture or IL-2 dependence. Suppressor T cells were not found in mice made tolerant by footpad injection of hapten-conjugated cells of the DETC line AU16, although they could be demonstrated in mice made tolerant by i.v. injection. Studies of lymphocyte proliferation in vitro suggested that hapten-conjugated AU16 cells may induce tolerance by inhibiting the proliferation of activated T lymphocytes.


Correlation between Ca(2+)-ATPase activity of rat islet cells and insulin secretion. Using medium with a low ionic strength, a low concentration of Ca2+ and Mg2+ and devoid of K+, we have measured Ca(2+)-ATPase activity in the homogenates of rat islets preincubated for 3 min with several hormones in the presence of 3.3 mmol glucose/l. Insulin secretion was also measured in islets incubated for 5 min under identical experimental conditions. Islets preincubated with glucose (3.3 mmol/l) and glucagon (1.4 mumol/l) plus theophylline (10 mmol/l), ACTH (0.11 nmol/l), bovine GH (0.46 mumol/l), prolactin (0.2 mumol/l) or tri-iodothyronine (1.0 nmol/l) have significantly lower Ca(2+)-ATPase activity than those preincubated with only 3.3 mmol glucose/l. All these hormones increased the release of insulin significantly. Dexamethasone (0.1 mumol/l) and somatostatin (1.2 mumol/l) enhanced the Ca(2+)-ATPase activity while adrenaline (10 mumol/l) did not produce any significant effect on the activity of the enzyme. These hormones decreased the release of insulin significantly. These results demonstrated that islet Ca(2+)-ATPase activity was modulated by the hormones tested. Their inhibitory or enhancing effect seemed to be related to their effect on insulin secretion; i.e. those which stimulated the secretion of insulin inhibited the activity of the enzyme and vice versa. Hence, their effect on insulin secretion may be due, in part, to their effect on enzyme activity and consequently on the concentration of cytosolic Ca2+. These results reinforce the assumption that Ca(2+)-ATPase activity participates in the physiological regulation of insulin secretion, being one of the cellular targets for several agents which affect this process.


Inhibitory action of somatostatin-14 on hormone-stimulated cyclic adenosine monophosphate induction in porcine granulosa and luteal cells. Somatostatin-14 (SRIF-14) inhibited, in a concentration-dependent manner, LH- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) induction in porcine granulosa and luteal cells. The inhibitory effect of SRIF-14 on hormone-induced cAMP generation was more potent in porcine ovarian cells than in the GH-3 pituitary cell line. The inhibitory effect of SRIF-14 was impeded by neutralizing its biological activity with specific antiserum. Preincubation of luteal and granulosa cells with phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-stimulated cAMP levels. SRIF-14 failed to inhibit LH- or forskolin-stimulated cAMP levels in cells preincubated with PMA. It is concluded that SRIF-14 inhibits hormone-stimulated cAMP induction in the porcine ovary. LH-induced protein kinase C activation may be physiologically important to alleviate the inhibitory effects of SRIF-14.


Suramin affects differentiated and undifferentiated human thyroid epithelial cells in vitro. In the last decade, suramin has become known for its antiproliferative, differentiation-inducing effects on cells and has been successfully used in the therapy of cancer patients. The present study was undertaken to investigate the effects of suramin on normal human thyroid cells in primary monolayer culture and to analyse whether it also affected cells from thyroid carcinomas. The results show that suramin, at concentrations similar to serum levels obtainable during therapy, inhibited the proliferation of thyroid cells as well as the secretion of thyroglobulin. It suppressed the activation of adenylyl cyclase in thyroid membranes and decreased the immunogenicity of the cells by reducing their surface expression of HLA-DR and ICAM-1. Although the morphology of differentiated thyroid cells remained unaffected by suramin, morphological changes compatible with differentiation were observed in cells from undifferentiated thyroid carcinomas when suramin was added to the culture medium. In conclusion, the data demonstrate that suramin has pronounced in-vitro effects on normal and neoplastic thyroid cells. It may, therefore, also be effective in patients with thyroid cancer, for whom no other form of treatment is available.


Pyridostigmine partially reverses dexamethasone-induced inhibition of the growth hormone response to growth hormone-releasing hormone. The GH response to insulin-induced hypoglycaemia and growth hormone-releasing hormone (GHRH) has been shown to be impaired in subjects with Cushing's syndrome and in healthy volunteers given oral glucocorticoids. Pyridostigmine is an anticholinesterase that stimulates GH secretion, probably by inhibition of hypothalamic somatostatin secretion. This work was designed to study the site of action of glucocorticoids in inhibiting the secretion of GH. Eight healthy male volunteers were studied on three occasions in random order. They took 2 mg oral dexamethasone or placebo at precisely 6-hourly intervals for 48 h before receiving 120 mg oral pyridostigmine or placebo, followed 60 min later by GHRH (100 micrograms) i.v. Samples for measuring GH were obtained at 15 min intervals for 2 h. The 'area under the curve' (AUC) for each of the treatments was significantly different: dexamethasone-pyridostigmine-GHRH (mean +/- S.E.M., 1938 +/- 631 mU/min per l), dexamethasone-placebo-GHRH (634 +/- 211) and placebo-placebo-GHRH (4267 +/- 1183) (P < 0.02, Wilcoxon test). In conclusion, dexamethasone given for 48 h significantly inhibited the AUC for GH following treatment with GHRH. However, pretreatment with pyridostigmine significantly reversed the inhibition although this was still partial. Our data suggested that this short-term suppressive effect of dexamethasone was independent of GHRH, and most probably relates to stimulation of the release of somatostatin.


The effects of glutamate agonists on voltage-clamped motoneurons of the lobster cardiac ganglion. The effects of L-glutamate and its analogues were studied in voltage-clamped motoneurons of the lobster cardiac ganglion. These excitatory amino acids caused a dose-dependent increase in membrane conductance and an inward current at the resting membrane potential. The EC50 for L-glutamate was 150 mumol 1(-1). The rank order of potencies of the various agonists was quisqualate greater than L-glutamate = L-aspartate greater than kainate greater than cysteine. Kainate, unlike the other agonists, showed no desensitization. Of various antagonists studied, only the quinoxalinediones inhibited the response to glutamate. These antagonists also reduced the amplitude and duration of the pacemaker-driven burst potential, suggesting that glutamate may be released by some of the endogenous synapses within the ganglion. The reversal potential of the glutamate-induced current was -15 mV. When Na+ was replaced with K+, the glutamate-induced current still reversed between 0 and -20 mV. When Na+ was replaced with the impermeant ion N-methyl-D-glucamine, the current was inhibited. The amplitude of responses evoked by glutamate and its analogues was reduced in salines containing either high or low concentrations of Ca2+. These results of pharmacological and of reversal potential and ion substitution experiments indicate that glutamate acts on receptors of the non-NMDA (N-methyl-D-aspartate), quisqualate/kainate type to open a channel permeable to both Na+ and K+.


Isolated left coronary ostial stenosis as the sole arterial involvement in Takayasu's disease. A 24-year-old woman with Takayasu's disease developed unstable angina pectoris. Angiographic studies demonstrated an isolated left coronary ostial stenosis without any other systemic arterial involvement. She is unique in that the coronary lesion, which is rarely the major manifestation of Takayasu's disease, is the sole arterial involvement of the disease.


Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL-12), is a 70-kD heterodimeric cytokine composed of two covalently linked chains, p40 and p35. NKSF/IL-12 has multiple effects on T and NK cells and was originally identified and purified from the supernatant fluid of Epstein-Barr virus (EBV)-transformed human B lymphoblastoid cell lines. We have produced a panel of monoclonal antibodies against both chains of NKSF/IL-12. Some of these antibodies have neutralizing activity, and several combinations of them have been used to establish sensitive radioimmunoassays detecting the free p40 chain, the free p35 chain, or the p70 heterodimer. Using these reagents, we have determined that most EBV-transformed human B lymphoblastoid cell lines constitutively produce low levels of the p70 heterodimer and an excess of the free p40 chain, whereas Burkitt lymphoma-derived, T, myeloid, and many solid tumor-derived cell lines produce neither. Production of both p40 and p70 is increased several-fold upon stimulation of the EBV-transformed cell lines with phorbol diesters. The ability of supernatant fluids from unstimulated and phorbol diester-stimulated cell lines to induce interferon gamma (IFN-gamma) production from T and NK cells, one of the effects of NKSF/IL-12, parallels the levels of production of the p70 heterodimer, known to be the biologically active form of NKSF/IL-12. Staphylococcus aureus Cowan I strain (SAC) and other stimuli induce accumulation of p40 mRNA and production of both p40 and p70 by peripheral blood mononuclear cells (PBMC). The producer cells appear to include both adherent cells and nonadherent lymphocytes, possibly B cells. The supernatant fluids from SAC-stimulated PBMC mediate the typical functions of NKSF/IL-12 (i.e., IFN-gamma induction, mitogenic effects on T/NK blasts, enhancement of NK cell cytotoxicity) at concentrations of p70 similar to those at which recombinant NKSF/IL-12 mediates the same functions. Moreover, these activities are significantly inhibited by anti-NKSF/IL-12 antibodies. The neutralizing anti-NKSF/IL-12 antibodies also inhibit 85% of the IFN-gamma production in response to SAC, an NKSF/IL-12 inducer, and approximately 50% of the IFN-gamma production in response to non-NKSF/IL-12-inducers such as IL-2, phytohemagglutinin, and anti-CD3 antibodies. These results indicate that induced or constitutively produced NKSF/IL-12 has a major role in facilitating IFN-gamma production by peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)


Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements. Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.


Challenges of space medical operations and life sciences management. The Kennedy Space Center (KSC) has been the premier launch and landing site for America's space program since the early 1960s. Visitors are cognizant of space vehicles, processing facilities and launch pads which are treasured national resources. However, most are unaware of the unique organization which supports launch and landing activities and manages the center's occupational medicine, environmental health, ecological and environmental monitoring functions, as well as human and plant research programs. Management of this multifaceted organization can be complex because funding its different functions comes from a number of sources. Additionally the diverse disciplines of personnel present a special challenge in maintaining professional competencies while assuring efficiency in cyclical operations. This article explains the organization's structure and reviews some of its accomplishments.


Controlled ecological life-support system. Use of plants for human life-support in space. Scientists and engineers within NASA are conducting research which will lead to development of advanced life-support systems that utilize higher plants in a unique approach to solving long-term life-support problems in space. This biological solution to life-support, Controlled Ecological Life-Support System (CELSS), is a complex, extensively controlled, bioengineered system that relies on plants to provide the principal elements from gas exchange and food production to potable water reclamation. Research at John F. Kennedy Space Center (KSC) is proceeding with a comprehensive investigation of the individual parts of the CELSS system at a one-person scale in an approach called the Breadboard Project. Concurrently a relatively new NASA sponsored research effort is investigating plant growth and metabolism in microgravity, innovative hydroponic nutrient delivery systems, and use of highly efficient light emitting diodes for artificial plant illumination.


An acetate-sensitive mutant of Neurospora crassa deficient in acetyl-CoA hydrolase. The predicted amino acid sequence of the product of the acetate-inducible acu-8 gene of Neurospora crassa, previously of unknown function, has close homology to the recently published sequence of Saccharomyces cerevisiae acetyl-CoA hydrolase. An acu-8 mutant strain, previously characterized as acetate non-utilizing, shows strong growth-inhibition by acetate, but will use it as carbon source at low concentrations. The mutant was shown to be deficient in acetyl-CoA hydrolase and to accumulate acetyl-CoA when supplied with acetate. As in Saccharomyces, the Neurospora enzyme is acetate-inducible.


Characterization of Aeromonas sobria TAP13 pili: a possible new colonization factor. Pili of Aeromonas sobria TAP13 were purified and characterized. The molecular mass of the pilin was estimated to be about 23 kDa by SDS-PAGE. The TAP13 pili were immunologically different from A. sobria Ae1 pili and A. hydrophila Ae6 W pili as previously reported, nevertheless all three had indistinguishable morphology and shared a high degree of homology in their N-terminal amino acid sequences. Strain TAP13 and its purified pili did not agglutinate human, rabbit or sheep erythrocytes. However, they adhered to rabbit intestine. Organisms pretreated with the Fab fraction of an antipilus antibody failed to adhere to rabbit intestine, and organisms did not adhere to intestine pretreated with purified pili. These results suggest that the pili are a colonization factor of A. sobria TAP13 for the rabbit intestine.


Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier. The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR. Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of spoIID. LytR is a 35 kDa protein which acts as an attenuator of the expression of both lytABC and lytR operons. Transcription of the lytABC operon proceeds from two promoters: PD, identified as P28-7 (Gilman et al., 1984), and an upstream PA. The former only is subject to LytR attenuation. Translational initiation of lytB and lytC is directed by UUG start codons, suggesting that lytA, B and C undergo coupled translation. Transcription of lytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to the fla regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the lytABC operon is discussed.


Are Sinc and the PrP gene congruent? Evidence from PrP gene analysis in Sinc congenic mice. Congenic mouse strains VM/Dk and VM-Sincs7/Dk differ at the Sinc gene, which controls the incubation period of scrapie in mice; VM/Dk mice are Sincp7p7 and VM-Sincs7/Dk mice are Sincs7s7. Restriction fragment length polymorphism and DNA sequencing analysis demonstrated that the PrP genes also differ in these strains, confirming the close genetic linkage of Sinc and PrP. Using the restriction enzyme HhaI, we have shown that at least 100 kb of DNA flanking the PrP gene differs between the two strains, and therefore the congruence of the Sinc and PrP genes is still not certain.


Potential significance of the cellular immune response against the macaque strain of simian immunodeficiency virus (SIVMAC) in immunized and infected rhesus macaques. The cellular immune response of seven rhesus macaques immunized with Tween-ether-treated macaque strain of simian immunodeficiency virus (SIVMAC) and three non-vaccinated control animals was investigated. Immunization elicited antigen-specific proliferating CD4+ cells in five of seven monkeys. Proliferating T cells were found in all animals protected from a first virus challenge. Cytotoxic T lymphocytes (CTLs) were not induced by the immunization. After the second challenge, the four formerly protected animals became infected, despite a strong proliferative CD4+ cell activity in three of them. All animals lost their proliferative activity 2 weeks after infection. After the first challenge four of the six infected animals exhibited a CTL response and after the second challenge, one of four newly infected macaques acquired a CTL response. The five animals with a CTL activity against SIVMAC proteins were protected from severe thrombocytopenia, which appeared in the five CTL-negative animals after infection. Our data show the induction of proliferative T cells by immunization with soluble SIVMAC antigen. This T cell reactivity was found in all animals protected from the first virus challenge, but did not confer protection from the second challenge. Interestingly, the proliferative T cell reactivity disappeared 2 weeks after virus infection. Furthermore a CTL response against viral proteins seems to protect infected animals from severe thrombocytopenia which is an early sign of AIDS in monkeys.


Serum antibodies to structural proteins of Hantavirus arise at different times after infection. An enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of serum antibodies against group-specific epitopes of the glycoproteins (G1, G2) and nucleoprotein (NP) of the genus Hantavirus. This assay was used to study the kinetics of the development of serum antibodies after natural infection with Puumala-like virus in humans. To this end a panel of 34 serum samples collected from individuals at different times after natural infection was tested by the ELISA. The samples were also tested for specific IgM and IgG levels against Puumala-like virus, which provided confirmatory data about the presumed timing of infection. It was shown that serum antibodies against the G1 epitope were present in the acute and early convalescent period just before antibodies to the NP epitope could be demonstrated. In contrast, antibodies to two G2 epitopes were present not earlier than in the convalescent and late convalescent period. Since all these categories of antibodies seem to persist for long periods, antibodies against the G1 epitope and the NP epitope may be of specific diagnostic value. Furthermore, levels of G1-specific antibodies and antibodies to either NP or G2 may allow estimation of the time elapsed following initial infection.


Evaluation of a new generation synthetic peptide combination assay for detection of antibodies to HIV-1, HIV-2, HTLV-I, and HTLV-II simultaneously. A new generation combination test (Detect-Plus, IAF BioChem, Montreal, Canada) based on synthetic peptides for HIV-1, HIV-2, HTLV-I, and HTLV-II was compared with three routine commercial screening assays and confirmatory assays to determine its sensitivity and specificity and to evaluate it as a substitute screening method. Samples from 356 sexually transmitted disease (STD) patients were tested by the four screening tests. All initially reactive samples were retested in duplicate by the corresponding EIA and repeatedly reactive samples were confirmed by Western blots for HIV-1, HIV-2, and HTLV-I/II. The confirmed positives detected by each screening assay were HIV-1 (23/356, 6.46%), HIV-2 (11/356, 3.09%), and HTLV-I/II (5/356, 1.4%). The new generation Detect-Plus test produced only two results (2/356, 0.56%) that were presumed to be false-positives in comparison to the screening tests, but the OD/CO values were just slightly high (1.5 and 1.9). There were no false-negative results, indicating that the sensitivity of the new combination test was excellent (100%). Compared with routine retroviral EIA assays, the test is easy to perform--the total time requirement is only 2 hr and there is no need for incubation equipment. The OD/CO values were very high when samples were positive, making even visual interpretation possible. We conclude that this new combination assay is an excellent screening method for detection of antibodies to the human retroviruses, and may be particularly useful for screening blood for transfusion and in epidemiological investigations.


Drug washout issues in studies of cerebral metabolism by positron emission tomography in psychiatric patients. Many studies of brain glucose utilization by positron emission tomography attempt to describe the modifications of the brain activity during psychiatric diseases. A major difficulty in such studies is the necessity to assess patients free of pharmacological treatment, in order to relate the measured changes in glucose utilization to the pathopsychology, and not to a drug effect. In this paper are reviewed the arguments from the literature allowing to estimate the drug washout time for considering the patients as drug-free. The review is focussed on the known effects of the psychotrops on brain glucose utilization. This time is approximately six months for the neuroleptics given orally, one month for antidepressants, and five and a half half-lives for benzodiazepines. Alternative research strategies for avoiding a long drug washout are mentioned, and ethical limitations are considered.


Quick blots and nonradioactive detection of DNA probes for the identification of mosquitoes. The quick blot protocol is an improved technique for preparing crude insect homogenates for hybridization to nucleic acid probes. Individual insects are ground in wells of a microtiter plate and transferred to a dot blot manifold. This allows preparation of multiple filters and provides uniformity and an orderly arrangement of samples. The high background detection resulting from use of crude insect homogenates with nonradioactive detection systems was eliminated by incubating quick blot filters in a laundry stain remover containing proteases. We used mosquito species-specific DNA probes to demonstrate the effectiveness of nonradioactive DNA labeling systems with quick blots.


Safety of Edhazardia aedis (Microspora: Amblyosporidae) for nontarget aquatic organisms. The susceptibility of common nontarget aquatic organisms to the microsporidium Edhazardia aedis was investigated in the laboratory. Eight predacious species along with 9 scavengers and filter feeders were tested. The nontarget organisms were not susceptible to infection by E. aedis and there was no appreciable mortality. To measure the relative safety of E. aedis to nontarget organisms, a simple mathematical expression was employed where risk is defined as the product of the probability of exposure and the result of exposure (infection) expressed as P(e)P(i). In these laboratory tests, the probability of exposure was fixed at 1 (maximum challenge) and the probability of infection was determined to be 0. Therefore, the risk associated with release of E. aedis into the environment is considered to be negligible under these conditions. The true risk for nontarget organisms to E. aedis can only be determined by careful evaluation of controlled field studies in the natural habitat of the target host.


Host range of Clostridium bifermentans serovar. malaysia, a mosquitocidal anaerobic bacterium. Clostridium bifermentans serovar. malaysia (C.b.m.) is toxic to mosquito larvae. In this study, we quantified its toxicity to the mosquitoes, Aedes aegypti, Ae. albopictus, Ae. caspius, Ae. detritus, Anopheles stephensi, An. gambiae, Culex pipiens and Cx. quinquefasciatus. Anopheles larvae are the most susceptible, followed by Ae. detritus and Ae. caspius, then Culex and other Aedes larvae. According to mosquito species, the LC50 varies from 7 x 10(3) to 1.3 x 10(6) cells/ml. Three concentrations (10(7), 10(6) and 10(5) cells/ml) of C.b.m., Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus were tested on Ae. aegypti, An. stephensi and Cx. pipiens larvae in order to determine the time necessary for each concentration to kill 50 and 90% of the population. Ninety percent of the 3 mosquito populations are killed within 4-15 h by the C.b.m. concentrations. Whatever the concentrations, C.b.m. kills at least 10 times less rapidly than B.t.i. but always quicker than B. sphaericus. Bioassays of C.b.m. bacterial cells or final whole culture were not toxic to Musca domestica and Drosophila melanogaster (Diptera) as well as to Phaedon cochleariae (Coleoptera) and Spodoptera littoralis (Lepidoptera).