Thyroid-stimulating antibodies and thyroid stimulation-blocking antibodies during the pregnancy and postpartum period: a case report.

This report describes a unique pattern of changes in thyroid function and thyroid antibodies in a woman during the course of two pregnancies and two postpartum periods. A 25-year-old woman developed hypothyroidism in the postpartum period after the delivery of her first child. She was found to have potent thyroid stimulation-blocking antibodies (TSBAb) in the serum. One year later, she became pregnant again, and during the pregnancy, TSBAb had decreased to an undetectable level. She gave birth to the second healthy child and developed postpartum thyrotoxicosis, probably due to destruction of the thyroid gland, which gradually resolved. In this postpartum period, serum TSBAb levels increased. Eight months postpartum, she developed what appeared to be Graves' disease with an elevated 123I-thyroid uptake. Serum thyroid-stimulating antibodies (TSAb) were found at that time, and the TSBAb had disappeared from her serum.


Chromogranin A as tumor marker in medullary thyroid carcinoma.

We measured plasma levels of chromogranin A (CgA) and calcitonin (CT) in 61 patients with surgically confirmed medullary thyroid carcinoma (MTC). CT was elevated in 46 patients, whereas CgA was elevated in 14 patients. Plasma levels of CgA and CT were moderately correlated (r = 0.87), but CgA became elevated in most patients only in advanced disease. Patients with high plasma CT values (greater than 10 micrograms/L) also had elevated CgA in 83% of cases. An elevated plasma CgA level despite normal CT levels was found in only 1 patient. In 8 MTC patients with moderately elevated basal CT levels, pentagastrin as a secretagogue usually was not able to release detectable amounts of CgA from MTC tissue. In 2 MTC patients, i.v. catheter sampling gave sharp gradients for CT concentrations (greater than 2.7-fold peak to peripheral ratios) and, therefore, precise MTC tissue localization, whereas no gradients were demonstrable for CgA (less than 1.2-fold). One patient with MTC and elevated CgA reached normal CgA plasma levels within 8 days after thyroidectomy. In metastatic tissue from 8 patients with MTC, CgA and CT were detectable immunohistologically in all cases, but plasma CgA was elevated only in 2 and CT in 7 of them. Plasma CgA levels in patients with MTC usually became elevated only in advanced disease and were not able to detect early disease stages, were correlated with CT levels, were not useful in stimulation tests or venous localization studies, and probably resulted from the release from MTC tissue as the major tissue source, as shown in the sporadic cases.


Thyroid growth immunoglobulins in feline hyperthyroidism.

Feline hyperthyroidism bears a strong clinical and pathologic resemblance to toxic nodular goiter in humans. To evaluate whether the observed thyroid growth might be due to circulating thyroid antibodies, as has been postulated in humans, we studied the effect of purified immunoglobulin (Ig) G preparations on a rat thyroid follicular (FRTL-5) cell line. When compared with control, hyperthyroid cat IgG caused significantly increased [3H]-thymidine (Tdr) incorporation into DNA (p less than 0.02) and stimulated cellular proliferation 15-fold. Stimulation of 3H-Tdr incorporation tended to be biphasic and could be inhibited completely by a potent, specific TSH receptor blocking antibody. Hyperthyroid cat IgG also significantly inhibited 125I-bTSH binding to porcine thyroid membranes, an effect that could be reproduced using electrophoretically pure IgG and normal cat thyroid membranes. Unlike its effect on growth, hyperthyroid cat IgG did not stimulate intracellular cAMP, and there was no correlation between thyroid function in vivo and IgG growth-promoting activity in vitro. These data suggest that elevated titers of thyroid growth IgGs, probably acting through the TSH receptor, are present in feline hyperthyroidism and may play a role in goiter formation. Unlike growth, the thyroid hyperfunction observed is not IgG dependent. Further study of feline hyperthyroidism may contribute important insights into human nodular goiter and into the mediation of thyroid growth in general.


Toxic Graves' disease with thyroid hemiagenesis: diagnosis using thyroid-stimulating immunoglobulin measurements.

Two patients with hemiagenesis of the thyroid gland experienced thyrotoxicosis. They constituted 1.1% of our clinic's total population of 178 thyrotoxic patients treated in the years 1986-1990 and 1.7% of 120 patients with thyrotoxic Graves' disease encountered during that period. The diagnosis was made on the basis of unilateral homogeneous 99mTcO4 uptake on thyroid scan, no change in the scan after both cessation of propylthiouracil (PTU) treatment for 4 days and TSH stimulation test, and high thyroid-stimulating immunoglobulin (TSI) levels. Both patients went into remission after PTU treatment, and TSI levels returned to normal. The diagnosis of toxic Graves' disease with thyroid hemiagenesis was, therefore, made. This combination is rare but important to recognize because treatment as well as prognosis might be different from that of toxic adenoma.


Fetal and neonatal hyperthyroidism and hypothyroidism due to maternal TSH receptor antibodies.

Autoimmune thyroid disease is a generic term that includes Graves' disease and Hashimoto's thyroiditis. In the former, there is overactivity of the thyroid due to the action of a thyroid-stimulating antibody (TSAb). Pathogenesis of Hashimoto's thyroiditis is largely cell-mediated immune destruction of the thyroid. Nonetheless, there may be either a goiter or an atrophic gland. There is evidence that in some patients the lack of goiter is associated with the presence in the blood of an antibody that inhibits the binding of TSH to its receptor. This TSH-binding inhibiting antibody (TBIAb), therefore, prevents TSH from stimulating the thyroid and constitutes an acceptable explanation for an agoitrous state. Collectively, TSAb and TBIAb, both of which are IgG, are known as TSH receptor antibodies (TRAb).


Analysis of chimerism after bone marrow transplantation using specific oligonucleotide probes.

DNA hybridization with synthetic oligonucleotide probes was used to follow 18 leukemia patients who received bone marrow transplantation from HLA-identical siblings. Five oligomers complementary to the tandem repetitive sequences of different hypervariable regions of human DNA were designed to produce simple restriction fragment length polymorphism patterns. Each probe hybridized to one or two bands in Hinf I-digested genomic DNA. Combined use of these probes enabled us to distinguish all sibling pairs. DNA analysis early post-transplant (15 days) detected donor-specific fragments in 14 of 18 subjects; two patients had a combination of recipient and donor fragments. Later post-transplant, (102-15 days), one of these two showed only recipient-specific fragments, and the other donor-specific fragments. These data are in accord with other markers of engraftment including cytogenetics and red blood cell phenotyping.


Epidermal growth factor selectively enhances NMDA receptor-mediated increase of intracellular Ca2+ concentration in rat hippocampal neurons.

We have previously reported that recombinant human epidermal growth factor (hEGF) facilitates induction of hippocampal long-term potentiation (LTP). In order to clarify the mechanism underlying the LTP-facilitating effect of hEGF, the influence of hEGF on intracellular Ca2+ concentration ([Ca2+]i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca2+]i were measured by microfluorometrically monitoring the fluorescence intensities from individual neurons loaded with fura-2. Application of hEGF (0.6-20 ng/ml) alone did not affect the basal level of [Ca2+]i in cultured hippocampal neurons, but significantly enhanced the [Ca2+]i increase induced by L-glutamate (3 x 10(-6) M). The N-methyl-D-aspartate (NMDA) (10(-5) and 3 x 10(-5) M)-induced [Ca2+]i increase was also enhanced by hEGF, but the quisqualate (10(-7) and 3 x 10(-7) M)-induced response was not affected by the presence of hEGF. These results suggest that hEGF selectively enhances the NMDA receptor-mediated responses in hippocampal neurons. This action of hEGF may underlie the facilitation of hippocampal LTP.


Immunohistochemical markers in rat cortex: co-localization of calretinin and calbindin-D28k with neuropeptides and GABA.

Calretinin and calbindin-D28k are two calcium-binding proteins which are present in separate populations of interneurons in cerebral cortex and hippocampus. To identify these cells with the populations expressing different transmitters, two-colour immunofluorescence was done with antibodies against the calcium-binding proteins plus antibodies against vasoactive intestinal peptide (VIP), somatostatin (SRIF), or gamma-aminobutyric acid (GABA). In neocortex, calretinin is partially co-localized with VIP (especially in the deeper layers) and is not co-localized with SRIF. Calbindin is largely co-localized with SRIF, and not with VIP. Both calretinin and calbindin are partially co-localized with GABA. In piriform and entorhinal cortex, the patterns resemble those in neocortex. In hippocampus, preliminary data indicate greater heterogeneity, especially in the ventral part; at least a few double-positive cells are present for every combination of calcium-binding protein and neuropeptide. These results expand the known diversity of local-circuit neurons in cortical regions.


Low levels of somatostatin-like immunoreactivity in neocortex resected from presumed seizure foci in epileptic patients.

The concentration of somatostatin-like immunoreactivity (SS-LI) was determined by radioimmunoassay in neocortical tissue resected from 20 patients with pharmacologically intractable complex partial seizures. Most resections included either the anterior temporal pole neocortex (15 cases) or cingulate gyrus neocortex (3 cases). The concentration of SS-LI was lowest in cortical tissue immediately adjacent to cortical tumors. Preoperative electrical recordings suggested that this tissue was the seizure focus. In vitro recordings showed that this tissue also exhibited abnormal hyperexcitable synaptic responses. Higher levels of SS-LI, similar to normal values previously reported in human cortex, were present in non-focal temporal neocortical tissue (resected from patients in whom the seizure focus was in the ipsilateral hippocampus) in which no hyperexcitable synaptic activity was present in vitro. The functional loss of inhibitory transmitters suggested by the low SS-LI levels might provide a theoretical basis for the hyperexcitability observed in vivo and in vitro.


GABAergic synaptic transmission in projections from the basal forebrain and hippocampal formation to the amygdala: an in vivo iontophoretic study.

We recorded extracellular responses from rat amygdaloid neurons in vivo after electrical stimulation of the basal forebrain and hippocampal formation. Iontophoretic application of the GABAA receptor antagonist, bicuculline, lead to the appearance of short latency evoked bursts after stimulation of either region. This occurred whether the baseline response was inhibitory or excitatory. Bicuculline only affected an early phase of inhibition, leaving a longer latency, longer duration phase unchanged or even increased. By contrast, the GABAB receptor antagonist, phaclofen, never produced such short latency evoked bursts. Both bicuculline and phaclofen increased the spontaneous rate of firing of amygdaloid neurons. The excitatory burst response to hippocampal formation stimulation of an amygdaloid candidate inhibitory neuron was blocked by CNQX (an antagonist of the AMPA subtype of glutamate receptor). Based on these and prior studies, it seems likely that the effects of hippocampal formation stimulation are mediated by feed-forward inhibition, in which GABAergic amygdaloid inhibitory neurons are excited by glutamatergic projections from the hippocampal formation. The effects of basal forebrain stimulation may be mediated by both feed-forward inhibition and direct, GABAergic inhibition.


Immunohistochemical markers in rat brain: colocalization of calretinin and calbindin-D28k with tyrosine hydroxylase.

Many dopaminergic cells of the substantia nigra are known to contain the calcium-binding proteins calretinin and calbindin-D28k. Catecholaminergic cell groups throughout the rat brain were therefore examined by two-colour immunofluorescence to determine whether they too contained these calcium-binding proteins as well as tyrosine hydroxylase (TH). Some TH+ cell groups are mostly positive for both calretinin and calbindin, notably in the ventral tegmental area, the interfascicular nucleus, and parts of the substantia nigra. Other TH+ cell groups in the midbrain, hindbrain and hypothalamus are very diverse; different cell groups are positive for calretinin, or calbindin, or both, or neither. In the olfactory bulb, entirely separate sets of periglomerular cells are positive for TH, calretinin and calbindin. However, there is considerable heterogeneity in calcium-binding protein expression within most cell groups, even in the substantia nigra. This could be a sign that calcium-binding proteins are regulated according to aspects of neuronal activity.


Food intake of lean and obese Zucker rats following ventricular infusions of adrenergic agonists.

The effect on food intake of adrenergic agonists administered into the third cerebral ventricle was studied in Zucker fatty and lean rats. The alpha 2 agonist, clonidine, produced a larger dose-related increase in food intake in lean rats than in the fatty rats. Dose-response curves show similar sensitivity, but decreased responsiveness in the lean animal. The beta 2 adrenergic agonist, salbutamol, produced similar food effect in obese and lean rats reducing food intake in the lean rats at the highest dose (300 nmol), and in the fatty rats at the two highest doses. The effects were small in both groups. The beta 3 agonist, BRL 37344, ([4-(2-((2-hydroxy-2-(3-chlorophenyl)ethyl)amino)-propyl)-phenoxy acetate]) produced a larger dose-related decrease in food intake in the fatty rat than in the lean rats. Dose-response curves showed that sensitivity of beta-receptors was similar, but the lean animals were less responsive. The beta-adrenergic blocking drug propranolol blocked the anorectic effect of BRL 37344 in the fatty rat. These studies suggest that in the fatty rat, the alpha 2 receptor system is tonically more active and the beta 3 receptor system tonically less active, a relationship that would explain the hyperphagia and development of obesity in these animals.


The infusion of an NMDA antagonist into perirhinal cortex suppresses amygdala-kindled seizures.

The seizure-modulating role of N-methyl-D-aspartate (NMDA) receptors located in several limbic areas was investigated. Amygdala-kindled rats were microinfused with the selective NMDA-receptor antagonist 2-amino-5-phosphonovalerate (APV, 1 microliter, 70 nmol) or artificial cerebrospinal fluid (ACSF) applied through a cannula located in either the amygdala or perirhinal, pyriform or deep prepyriform cortices. APV infused into the stimulation site raised the threshold for seizure generation. Surprisingly, APV infused into perirhinal cortex, but not into other regions, also dramatically suppressed behavioural seizures and afterdischarges (AD) elicited 5 min after the infusion. If stimulus intensities were markedly elevated however, the seizure suppression was overcome. This latter effect was reversible and repeatable, as seizures and AD were reliably reinstated when these animals were stimulated after infusion with ACSF. A similar effect, whereby perirhinal infusions blocked seizure activity, was also demonstrated in an animal kindled from the olfactory bulb and in one kindled from the perforant path. These results suggest that NMDA receptors located in the perirhinal cortex may play a major role in the modulation of AD activity elicited from more distal brain regions. Furthermore, activation of perirhinal cortex may be a critical requirement for the generation of amygdala-stimulated AD in the kindled animal.


Effects of immunoneutralization of dynorphin1-17 and dynorphin1-8 on the activity of central dopaminergic neurons in the male rat.

The effects of administration of antibodies against dynorphin1-17 (DYN1-17-AB) and dynorphin1-8 (DYN1-8-AB) were examined on the activity of dopaminergic (DA) neurons comprising the nigrostriatal, mesolimbic, tuberoinfundibular and periventricular-hypophysial systems in the male rat brain. DA neuronal activity was estimated by measuring the concentration of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in brain (striatum, nucleus accumbens, median eminence) and pituitary regions (intermediate lobe) containing terminals of these neurons. The intracerebroventricular administration of either DYN1-17-AB or DYN1-8-AB produced a time-related increase in the activity of tuberoinfundibular and periventricular-hypophysial DA neurons, but failed to alter the activity of nigrostriatal or mesolimbic DA neurons. The ability of both DYN1-17-AB and DYN1-8-AB to enhance the activity of tuberoinfundibular and periventricular-hypophysial DA neurons was reversed by the kappa opioid agonist U-50,488. These results indicate that DYN1-17-AB and DYN1-8-AB, presumably by binding endogenous dynorphins, remove a tonic inhibitory action of these opioid peptides on tuberoinfundibular and periventricular-hypophysial DA neurons.


Involvement of central dopamine and D1 receptors in stress-induced colonic motor alterations in rats.

The role of central versus peripheral influence of dopamine (DA) in the genesis of emotional stress (ES) induced by fear to receive electric footshocks on colonic motility was evaluated in rats equipped with implanted electrodes on the proximal colon. In control rats, the frequency of colonic spike bursts increased from 7.5 +/- 1.9 to 16.0 +/- 2.1 per 10 min when the rats were placed in a test box where they had previously received electric footshocks. This increase induced by ES was significantly p less than 0.05, reduced by previous ICV or IP administration of (+)SCH 23390 (a D1 receptor antagonist) at doses of 10 and 100 micrograms/Kg, respectively. Although sulpiride (a D2 antagonist) injected ICV or IP at similar doses had no effect on the ES-induced increase in the frequency of colonic spike bursts. DA (100 micrograms/kg), and the selective D1 (SKF 38383) or D2 (quinpirole) receptor agonist injected ICV at a dose of 5 micrograms/kg also increased significantly by 48.7, 54.8, and 68.7%, respectively, the colonic spike burst frequency whereas they are inactive when injected IP at similar and higher doses. These results suggest that, in rats, (a) emotional stress stimulates colonic motility through the stimulation of dopaminergic neurons involving D1 receptors and (b) exogenous activation of central D1 and D2 receptors similarly stimulate colonic motility by increasing the occurrence of colonic spike bursts.


Influences of gender, gonadectomy, and estrous cycle on GABA/BZ receptors and benzodiazepine responses in rats.

Benzodiazepines (BZ) and steroid hormone derivatives can potentiate the inhibitory actions of GABA through interactions with the GABAA/BZ/chloride ionophore complex. The present study examines whether the in vivo hormone milieu of rats modulates GABA/BZ receptors and/or benzodiazepine responses. The influences of gender, estrous cycle, and the diminution of steroid levels on GABA/BZ receptors and BZ anticonvulsant responses were tested by comparing these parameters in groups of intact male, intact female, orchidectomized, and ovariectomized rats. The hormonal milieu appears to modulate the GABA recognition site and possibly GABA-related responses in rats. This is evidenced by the decrease in cortical GABAA receptor affinity seen in females compared with other hormone groups and the gender-related difference observed in susceptibility to seizures induced by the GABA antagonist bicuculline. In cycling females, high circulating levels of progesterone were correlated with heightened seizure thresholds, suggesting that progestins serve a protective role in the control of seizure activity. Although a gender-related difference in cortical BZ binding affinity was observed, BZ receptor parameters in several other brain areas and BZ anticonvulsant responses were unaffected by physiological fluctuations in gonadal hormones.


NMDA receptors mediate the behavioral effects of amphetamine infused into the nucleus accumbens.

The present experiments examined glutamate-dopamine interactions within the nucleus accumbens in rats. It has been hypothesized that dopaminergic nerve terminals exert a modulatory influence on glutamate-mediated signals from corticolimbic areas. In the present studies, the effect of the selective NMDA (n-methyl-d-aspartate) antagonist AP5 (2-amino-5-phosphonopentanoic acid) on amphetamine-mediated behaviors was observed. In two behavioral paradigms, AP5 (0, 0.05, 0.5, 1.0 micrograms bilaterally) was microinjected immediately prior to amphetamine (5 micrograms bilaterally) in the nucleus accumbens. In the first experiment, the influence of AP5 on amphetamine-induced motor activity was examined. AP5 dose-dependently reduced the effectiveness of amphetamine in stimulating motor behavior. AP5 alone, paradoxically, tended to increase motor activity. In the second experiment, the effects of AP5 on amphetamine-potentiated responding (lever pressing) for conditioned reward (CR) were investigated. Normally, when amphetamine is infused into the nucleus accumbens, a marked potentiation of CR responding occurs. Prior infusion of AP5 also attenuated this behavioral effect of amphetamine. The results demonstrate that NMDA receptors within the nucleus accumbens mediate the behavioral consequences of increased dopamine release. They provide additional evidence for the involvement of limbic-striatal connections in the activating and reinforcing effects of psychostimulant drugs.


[The effect of BL 343 Ac, a beta-adrenolytic, on the cervical ganglion].

The activity of the newly synthetized substance BL 343 Ac (propyl-3-acetyl-4[/2-hydroxy-3-isopropyl amino/propoxy] carbanylate of hydrochloride), a beta 1-adrenolytic with sympathomimetic activity was investigated by means of the electrophysiological method of sucrose gap. Its effect on the membrane potential of a population of neurons of the superior cervical ganglion (GCS) of the cat was analyzed with the aim to contribute to the knowledge on the selectivity of action of this substance. In the GCS of the cat, which contains predominantly beta 2-adrenoceptors, BL 343 Ac given in concentrations from 0.01 to 50 mumol.l-1 and after single application into the superfusion stream close to the ganglion in doses from 0.01 to 1,000 nmol did not induce an unequivocal change in the membrane potential or a response comparable to that elicited by isoprenaline. No blocking effect of the substance BL 343 Ac (0.01-50 mumol.l-1) was observed on isoprenaline induced depolarization of the cat GCS. The obtained results indicate that BL 343 Ac fails to exert either beta 2-adrenomimetic or beta 2-adrenolytic effect on the GCS of the cat. (Fig. 2, Ref. 11.)


[The delta F508 mutation which causes cystic fibrosis and its association with closely linked DNA polymorphisms in the Slovak population].

Linkage relationships between DNA polymorphism metH/TaqI as well as KM19/PstI and the mutation causing cystic fibrosis (CF) were analyzed in 48 families from Slovakia with th occurrence of CF. The polymorphism metH/TaqI did not show linkage disequilibrium with CF mutation. A pronounced allelic association was however found between CF mutation and KM19/PstI polymorphism. Of the 83 CF chromosomes analyzed, the given mutation was associated with the 6.6 kb allele in 82% of cases, while the rate of this allele in chromosomes without the mutation amounts only to 24%. The value of the standardized disequilibrium coefficient SCD = 0.58. Delta F508 deletion was addressly studied in 25 patients (i.e. 50 CF chromosomes). Of the 50 CF mutations, the given deletion was in 64% (32), while the remaining 36% (18) of mutations were of other, closely not identified types. Delta F508 deletion is in marked allelic association with the 6.6 kb allele of KM19/PstI polymorphism (SCD = 0.68). Between the given allele of KM19/PstI polymorphism and CF mutation no other allelic association was found but with delta F508. (Tab. 6, Fig. 1, Ref. 18).


[Multidrug resistance and the P-glycoprotein].

A survey is presented on the information concerning the nature and molecular mechanism of multi drug resistance, a phenomenon involving the resistance of tumor cells to different types of chemotherapeutic agents. P-glycoprotein is by its enzymatic activity directly responsible for expelling xenobiotics from the intracellular space and thus also for the development of MDR. Its detection provided new possibilities for causal studies of this type of resistance as well as for its in vitro modelling. In the presented survey, the function of P-glycoprotein is characterized also in cells of normal nontumorous tissue.


CLIP-170 links endocytic vesicles to microtubules.

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BIK1 proteins. These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.


Medication-induced systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease affecting a variety of tissues and organs. The diagnosis of SLE can be made only after several related illnesses are considered and ruled out. The etiology of SLE is unclear, but hormonal factors, environmental toxins, infectious viruses, genetic predisposition, and certain medications have all been considered risk factors. Idiopathic SLE is seen predominantly in young women, with a female:male ratio of approximately 10:1. Each patient is unique and may suffer from a variety of signs and symptoms. The disease is highly unpredictable, and most patients experience flare-ups or fluctuations. The epidemiologic characteristics of medication-induced SLE (MI-SLE) are different from those of idiopathic SLE. Musculoskeletal symptoms predominate the clinical presentation of MI-SLE, while renal and central nervous system involvement is rare or absent. Moreover, a greater percentage of caucasian patients with no female predominance is evidenced in MI-SLE. Several medications can produce positive results on an antinuclear antibody test with or without evidence of clinical lupus. Hydralazine and procainamide are the most commonly recognized medications for inducing SLE. The onset of procainamide- and hydralazine-induced SLE occurs after 50 years of age, which is directly related to the age of the population using these medications. Estrogen-containing oral contraceptives and ibuprofen can exacerbate the symptoms of idiopathic SLE. Clinical judgment dictates the importance of careful patient monitoring and selection of therapy.


Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53.

Gene amplification occurs at high frequency in transformed cells (10(-3)-10(-5)), but is undetectable in normal diploid fibroblasts (less than 10(-9)). This study examines whether alterations of one or both p53 alleles were sufficient to allow gene amplification to occur. Cells retaining one wild-type p53 allele mimicked the behavior of primary diploid cells: they arrested growth in the presence of drug and failed to demonstrate amplification. Cells losing the second p53 allele failed to arrest when placed in drug and displayed the ability to amplify at a high frequency. Thus, loss of wild-type p53 may lead to amplification, possibly caused by changes in cell cycle progression. Other determinants can by-pass this p53 function, however, since tumor cells with wild-type p53 have the ability to amplify genes.


V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes.

Lymphoid cells from scid mice initiate V(D)J recombination normally but have a severely reduced ability to join coding segments. Thymocytes from scid mice contain broken DNA molecules at the TCR delta locus that have coding ends, as well as molecules with signal ends, whereas in normal mice we previously detected only signal ends. Remarkably, these coding (but not signal) ends are sealed into hairpin structures. The formation of hairpins at coding ends may be a universal, early step in V(D)J recombination; this would provide a simple explanation for the origin of P nucleotides in coding joints. These findings may shed light on the mechanism of cleavage and suggest a possible role for the scid factor.


Thialysine-resistant mutants and uptake of lysine in Schizosaccharomyces pombe.

Mutants defective in lysine transport were isolated and characterized. After UV-mutagenesis colonies resistant to thialysine, a toxic analogue of lysine, were isolated and L-lysine uptake into the mutant strains was analyzed. Among the thialysine-resistant strains a group of mutants was found, where the half-saturation constant, KT, of the high-affinity transport system for lysine was higher than in the wild-type, the high-affinity transport system for basic amino acids being specifically affected. This was confirmed by a complementation test in which all the thialysine-resistant strains with a higher KT for lysine uptake belonged to one complementation group. Kinetic and genetic analysis showed that our mutants were identical with can1-1 mutants, showing that a single high-affinity system for the transport of basic amino acids exists in S. pombe.


Novel mitochondrial genomes in Brassica napus somatic hybrids.

The mitochondrial genomes of nine male-fertile and two Ogura cytoplasmic male-sterile (cms) Brassica napus somatic hybrids were probed with 46 mitochondrial DNA fragments. The distribution of information obtained from each fusion partner was not random. Several regions, including the coxI gene and a major recombination repeat sequence, were always derived from the Brassica campestris fusion partner, and some regions were always derived from the Ogura mitochondrial genome. Novel fragments occurred in seven distinct regions. Some of the rearrangement breakpoints were located near the evolutionary breakpoints relating the mitochondrial genomes of the Brassica species. The sizes of the mitochondrial genomes in the somatic hybrids ranged from 224.8 to 285.3 kb. A direct correlation between a specific gene and the cms phenotype was not observed; however, a possible cms-associated region was identified. It corresponds to a region that was identified through analysis of fertile revertants from a cms B. napus cybrid.


[Evoked visual responses in schizophrenic patients].

The authors investigated during prolonged ambulatory treatment with the neuroleptic preparation isofloxythepine (IFT) changes of visually induced responses in 24 psychotic patients. The reference group was formed by 10 healthy volunteers examined before and after administration of a single dose of IFT by the oral route. In addition to the visually induced responses the authors investigated also plasma prolactin levels (PRL). It was found that the latency of the P1 peak and amplitude between peaks A1 are in psychotic patients altered, as compared with healthy subjects. The latency of the P1 peak is after a single dose of IFT in healthy subjects longer and the amplitude A1 higher than in the sick group. In healthy subjects after administration of IFT a negative correlation was observed between the amplitude and the basal PRL level. In patients no relationship was found between PRL levels and parameters of the visually induced response.


Powder coating mixture of a novel anxiolytic, Y-23684: dissolution characteristics and bioavailability.

A powder coating mixture was investigated with a view toward improving the dissolution property of the anxiolytic 2-(4-chlorophenyl)-5,6-dihydro-[1]benzo-thiepino-[5,4-c]-pyrida zin-3(2H)-one 7-oxide (1), which was barely water soluble. The powder coating mixture in various ratios of 1 and cornstarch was prepared in an automated mortar. Among these mixtures, at the optimum ratio of 1 and cornstarch (2:1, 67% drug content), the powder coating mixture gave a maximized effect for solubilizing 1 on the bases of stability and solubility. Conventional granules were made from the 67% powder coating mixture. The granules showed an excellent absorption profile in beagle dogs. The mechanism of the solubilizing effect resulting from a pharmaceutical process was also discussed.


International poster parade: sight bites from the 18th World Congress of Dermatology New York City June 12 to 18, 1992.

Most of what we learn comes from reading, not from listening. Thus, for us, the poster presentation is ideal. It allows us to escape from the confinement of the lecture hall and from the constraint of having to listen to the obvious, the repetitious, the uninteresting, and the irrelevant. Come with us and scan those posters that caught our roving clinical eye as we viewed a thousand poster "lectures."


Partial release of aminopeptidase N from larval midgut cell membranes of the silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C.

1. The membrane anchor of aminopeptidase N associated with larval midgut cell membranes of the silkworm, Bombyx mori, was investigated by using phosphatidylinositol-specific phospholipase C (PIPLC) and proteases. 2. Aminopeptidase N, which was virtually all localized in the brush border membrane, was solubilized by PIPLC but not by papain or trypsin. 3. Detergent-solubilized amphiphilic aminopeptidase N was converted into a hydrophilic form by PIPLC but not by papain. 4. Either of these effects of PIPLC on aminopeptidase N was maximally 40%. 5. These results suggest that in larval midgut cells of the silkworm, B. mori, at least 40% aminopeptidase N is anchored in the brush border membrane via glycosyl-phosphatidylinositol.


Clinical applications of cytometry: 6th annual meeting.

The Sixth Annual Clinical Applications of Cytometry Meeting was held September 11-14, 1991, in Charleston, SC. Attendance reached a record 470. The meeting provides a forum for interactions among investigators who utilize cytometry as a tool in their clinical immunology, cell biology, hematology, and cancer investigations. Clinical laboratory directors and their technical staff find the meeting of practical value because of the presentation of new applications that they can take home to their own laboratories. The emphasis of the meeting is on advances in the application of cytometry to clinical problems. Often, advances result from new dyes or reagents or improved instrumentation. Sometimes they result from advances in biology that make the studies possible. Occasionally a new way of looking at the same data provides a useful answer. In every case, the effort is to provide a reliable, straightforward way to quantitate biologic information in order to provide improved diagnosis or treatment of human disease.


Comparison of strategies to detect and quantitate uniquely marked cells in intra- and inter-species hemopoietic chimeras.

Evaluation of the outcome of successful bone marrow transplantation and indepth studies of transplantation biology rely increasingly upon detection and enumeration of donor hemopoietic cells in the transplanted recipients. The ability to detect and enumerate low levels of donor engraftment in interphase cell subpopulations in hemopoietic chimeras is particularly important for studies of mixed lineage chimerism, early relapse manifestations, and engraftment of subpopulations present at low frequency. We describe and compare the sensitivity and specificity of DNA-based detection strategies (fluorescence in situ hybridization, in vitro DNA amplification using the polymerase chain reaction) and flow cytometric analysis of cell surface markers to detect cells carrying marker DNA or proteins in syngeneic (mouse-to-mouse) and xenogeneic (mouse-to-human, monkey, sheep) backgrounds. DNA-based detection strategies offer advantages of rapid analysis and enumeration of target cell frequencies with detection sensitivities approximating 10(-4). The sensitivity of immunofluorescence-linked flow cytometric-based detection of nucleated leukocytes approached 10(-3), whereas flow cytometric-based detection of fixed human erythrocytes was feasible at cell frequencies of 10(-5). Data described in this manuscript should facilitate selection of appropriate methodologies for assessment of hemopoietic chimerism following transplantation.


[Hantavirus infection with acute kidney failure].

Hantavirus infection was confirmed by history, symptoms and biochemical changes, as well as immunofluorescence test in 29 patients (24 men, 5 women; mean age 36.9 +/- 11.5 years) with nontraumatic renal failure (ANF), retrospectively in 15 patients. Cardinal symptoms were acute onset (n = 29), fever (n = 27), pain in the flanks, abdomen or head (n = 27), reduced glomerular filtration rate (n = 29), proteinuria (n = 25) and thrombocytopenia (n = 16). Normal renal function was restored in all patients. Follow-up examination of 15 patients 6-7 years after the acute illness revealed normal blood pressure, normal serum creatinine, absent proteinuria and normal inulin clearance in all, thus confirming the favourable prognosis of the infection in Western Europe. Nonetheless, because Hantavirus infection is by no means rare, it should be included in the differential diagnosis of acute renal failure.


Effect of the cyclopyrrolones suriclone and RP 59037 on body temperature in mice.

The effects of the cyclopyrrolones suriclone and RP 59037 on body temperature were investigated in male TO mice. The full agonist suriclone (3, 10, 30 mg/kg i.p.) produced significant hypothermia which was inhibited by concurrent administration of benzodiazepine receptor antagonists of both benzodiazepine (flumazenil; 10 mg/kg i.p.) and beta-carboline (ZK 93426; 3 mg/kg i.p.) structure. The response to suriclone (10 mg/kg i.p.) was also attenuated by benzodiazepine (Ro 17-1812; 10 mg/kg i.p.) and beta-carboline (ZK 91296; 30 mg/kg i.p.) partial agonists - which have no effect on body temperature per se. In contrast with these compounds, the cyclopyrrolone partial agonist RP 59037 (10, 30 mg/kg i.p.) produced significant hypothermia itself (although it was much less efficacious in this respect than the full agonist) and at a dose of 30 mg/kg failed to block the decrease in body temperature induced by suriclone (10 mg/kg i.p.). Thus suriclone acts as a full agonist at benzodiazepine receptors in the body temperature paradigm. RP 59037 possesses some partial agonist properties in this model, however, it appears to have greater intrinsic activity than other partial agonists tested previously.


Tolerance to hypoactivity and sensitization to hyperactivity after chronic treatment with a presynaptic dose of lisuride in rats.

We studied the adaptive changes of the locomotor effects of lisuride, a selective agonist for dopamine (DA) D2 receptors, and the functional state of D1 and D2 receptors after repeated administration of lisuride at a dose supposed to act preferentially on DA autoreceptors. Rats were treated daily with saline or lisuride, at a dose that causes a significant reduction in locomotor activity when given to naive rats (25 micrograms/kg i.p.), for 33 days and the effect of different challenging doses of the drug on locomotor activity was measured at different times during and after the treatment. The functional state of D1 and D2 DA receptors was evaluated by measuring SKF 82526-stimulated and LY 171555-inhibited adenylate cyclase (AC) activity in the caudatus/putamen, nucleus accumbens and substantia nigra and naive and chronically treated rats. There was a progressive decline in the ability of lisuride to decrease locomotor activity in rats given daily injections of lisuride, and there was a marked reduction in the threshold dose of lisuride for causing hypermotility. The functional state of DA receptors, positively or negatively linked to AC activity, was not modified by the treatment. The most suitable explanation of the reported adaptive behavioral changes is a down-regulation of DA autoreceptors after chronic treatment with presynaptic doses of lisuride.


Chaperoning practices of Ohio family physicians.

The medical literature contains no consistent recommendations regarding chaperon practices during physical examination of patients. The objective of this study was to determine the chaperon practices of Ohio family physicians. A questionnaire was mailed to all 1,786 active members of the Ohio Academy of Family Physicians. The questionnaire solicited information about the physicians' chaperon practices during physical examinations of the rectum, sexual and genital organs, and the female chest. The response rate was 74.6%. During female genital examinations, chaperons were used by 79.4% of male and 31.9% of female family physicians. Only 1.4% of male and 14.4% of female physicians used chaperons during male genital examinations. Chaperon practices were influenced by the gender of the physician and patient, the type of examination, and the availability of an assistant. No standard of care exists regarding chaperon practices among Ohio family physicians.


[Rectal ganglioneuromatosis and multiple type IIb endocrine neoplasia].

Intestinal tract ganglioneuromatosis was discovered in a 27-year old man through surgical biopsy specimens obtained after acute intestinal obstruction. The patient then developed type IIb endocrine neoplasia including a marfanoid morphotype, mucosal neuromatosis, metastatic medullary carcinoma of the thyroid and bilateral pheochromocytoma. The histological and clinical features, the pathophysiological mechanisms of intestinal ganglioneuromatosis and its place within the intricate group of neurocristopathies are discussed.


The effect of dopamine on rat gastric motility.

The inhibitory mechanism of dopamine (DA) on rat gastric motility was investigated in association with DA receptors. Gastric movement was assessed according to the method of Jacoby et al and was expressed with the system of Ludwick et al. (1968). DA inhibited gastric movement in both the corpus and antrum in a dose-dependent manner. Domperidone, a specific antagonist of DA2 receptor, suppressed DA-induced inhibition of gastric movement in a dose-dependent manner. SCH23390, a specific antagonist of DA1 receptor did not affect DA-induced inhibition of gastric movement. LY171555, a specific agonist of DA2 receptor, inhibited gastric movement in both the corpus and antrum in a dose-dependent manner. SKF38393, a specific agonist of DA1 receptor, did not affect gastric movement. These results indicate that DA plays an important role in the inhibitory regulation of gastric motility, through DA2 receptor but not DA1 receptor.


Biochemical and serological characterization of Escherichia coli fimbrial antigen F165(2).

Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves. A fimbrial component with an M(r) of 17,200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E. coli strain 4787 of serogroup O115. This fimbrial component of F165 antigen was named F165(2). Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic. The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae. The amino acid sequence was determined for approximately 40% of the molecule. For the first 33 residues, the F165(2) sequence was identical to that of F1B fimbriae and very similar to that of F1C. Fimbriae F165(2) could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera.


Recent amplification of triose phosphate isomerase related sequences in lettuce.

A random cDNA clone was identified as distinguishing near-isogenic lines for downy mildew resistance in lettuce. The clone detected multiple restriction fragments in genomic Southern blots of lettuce. Restriction fragment length polymorphisms (RFLPs) detected by this clone mapped to separate clusters of resistance genes; therefore, these sequences were studied in a greater detail. Sequence analysis indicated that the cDNA encoded the glycolytic enzyme triose phosphate isomerase (TPI). The lettuce clone shares 85% sequence similarity at the amino acid level with TPI from maize. TPI-related sequences were mapped in lettuce using three crosses. Ten loci were distributed in six linkage groups. Possible mechanisms of amplification and dispersion were investigated. Retrotransposition was excluded, since intron five is retained in all TPI-related genomic sequences. Large scale chromosomal rearrangements were not involved, as RFLP markers flanking TPI loci were not duplicated. A high level of genomic variability was detected by the TPI clone; 37 different restriction fragments were detected in Southern hybridizations to 64 populations of lettuce including 47 cultivars of Lactuca sativa and five wild species. Species distantly related to L. sativa had few TPI loci, indicating that their amplification and dispersion were recent and had occurred after the emergence of the L. serriola complex.


Thymopentin reduces the susceptibility of aged mice to cutaneous leishmaniasis by modulating CD4 T-cell subsets.

BALB/c mice are highly susceptible to Leishmania major infection. The susceptibility increases progressively with the age of the mice. Aged mice produce progressively lower levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) but higher levels of IL-4 compared to younger mice. Thymopentin, a pentapeptide with thymopoietin activity, dramatically increases the resistance to Leishmania major infection in aged mice. The thymopentin-treated mice produce enhanced levels of IL-2 and IFN-gamma, but significantly reduced amounts of IL-4. Thus, it appears that the age-related susceptibility to cutaneous leishmaniasis is correlated with the enhancement of Th2 and the reduction of Th1 cell activities. Furthermore, thymic hormone may play an important role in the induction and function of these two subsets of CD4 T cells.


The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.


Inhibition of T-cell mediated cytotoxicity by Novobiocin suggests multiple pathways for both CD4+ and CD8+ cytotoxic T cells.

The effect of the topoisomerase II inhibitor Novobiocin on T-cell mediated cytotoxicity was tested under various assay conditions. When effector cells were class I major histocompatibility complex (MHC)-specific CD8+ cytotoxic T cells (Tc), Novobiocin caused a biphasic pattern of inhibition and the two components of the inhibition could be separated based on Ca2+ requirement. Unseparated populations of class II MHC specific Tc, containing CD4+ and CD8+ effectors gave the same pattern of inhibition. When CD8+ cells were depleted from the latter population of effectors, different patterns of inhibition from those obtained with CD8+ Tc were seen and furthermore the target affected the pattern of inhibition. Overall the results add further support to there being more than one pathway of CD8+ T-cell mediated cytotoxicity and further illustrate differences between CD4+ and CD8+ T-cell mediated cytotoxicity.


Marked dendritic cell-T cell cluster formation in the pancreatic lymph node of the non-obese diabetic mouse.

Dendritic cells (DC) isolated from various lymph node (LN) groups of pre-diabetic non-obese diabetic (NOD) (4-20 weeks of age) and age-sex-matched control mice were analysed for their surface antigen phenotype and their ability to cluster lymphocytes. The draining LN of the pancreas (PLN) of 8-week-old NOD mice with active autoimmune disease were significantly enlarged in comparison to the axillary LN of the same NOD mice and the PLN from control mice. NOD DC isolated from PLN and other LN demonstrated classical DC morphology, were highly major histocompatibility complex (MHC) class II antigen positive, and were 50-70% 33D1+ (DC-specific antibody). In an assay for DC-T cell clustering, DC from the PLN of 8-20-week-old NOD formed large clusters (greater than 10 cells) with PLN cells at a frequency three to 20 times greater than that observed with DC and LN cells from the PLN of 8-week-old control mice, the PLN of 4-week-old NOD mice, and axillary/inguinal LN of 8-week-old NOD mice. Clustered cells were 80% Thy-1.2+ (56% L3T4, 17% Lyt-2+). Specificity of clustering was demonstrated as PLN DC clustered only PLN T cells in the assay; axillary/inguinal (A/I) DC added to PLN LC did not induce clustering nor did PLN DC induce clustering of the A/I population. Cell proliferation in isolated PLN DC/LC clusters was markedly greater than that of A/I clusters and of non-clustered PLN cells. These data demonstrate that DC from the PLN of NOD mice with active autoimmune disease form stable clusters with T cells from the PLN and these clusters are the major source of proliferating T cells in these LN. We hypothesize that PLN DC may play an important role in the autoimmune disease of the NOD mouse.


Ancestral haplotypes reveal the role of the central MHC in the immunogenetics of IDDM.

The major histocompatibility complex (MHC) contains multiple and diverse genes which may be relevant to the induction and regulation of autoimmune responses in insulin dependent diabetes mellitus (IDDM). In addition to HLA class I and II, the possible candidates include TNF, C4, and several other poorly defined polymorphic genes in the central MHC region. This study describes two approaches which take advantage of the fact that the relevant genes are carried by highly conserved ancestral haplotypes such as 8.1 (HLA-B8, TNFS, C4AQ0, C4B1, DR3, DQ2). First, three "diabetogenic" haplotypes (two Caucasoid and one Mongoloid) have been compared and it has been shown that all three share a rare allele of BAT3 as well as sharing DR3, DQ2. In 43 sequential patients with IDDM the cross product ratio for BAT3S was 4.8 (p less than 0.01) and 6.9 for HLA-B8 plus BAT3S (p less than 0.001). Second, partial or recombinant ancestral haplotypes with either HLA class I (HLA-B8) or II (HLA-DR3, DQ2) alleles were identified. Third, using haplotypic polymorphisms such as the one in BAT3, we have shown that all the patients carrying recombinants of the 8.1 ancestral haplotype share the central region adjacent to HLA-B. These findings suggest that both HLA and non-HLA genes are involved in conferring susceptibility to IDDM, and that the region between HLA-B and BAT3 contains some of the relevant genes. By contrast, similar approaches suggest that protective genes map to the HLA class II region.


Polymorphic analysis of the three MHC-linked HSP70 genes.

Three genes encoding members of the M(r) 70,000 heat shock protein family (HSP70) are known to lie in the class III region of the human major histocompatibility complex. In order to determine whether these genes or their protein products exhibit any polymorphism the three genes have been specifically amplified from genomic DNA and sequenced. The HSP70-1 and HSP70-2 genes encode the major heat-inducible HSP70. A comparison of the nucleotide sequences of these genes from B8, SC01, DR3, B18, F1C30, DR3, and B7, SC30, DR2 haplotypes has revealed only very limited sequence variation which is not associated with any amino acid polymorphism. The HSP70-Hom gene encodes a protein that is highly related to HSP70-1, but which is not heat-inducible. Nucleotide sequence analysis of this gene from different haplotypes has revealed a Met----Thr amino acid substitution at residue 493 in a number of the haplotypes tested. This variable amino acid lies in the proposed peptide-binding site of the HSP70-Hom protein.


Involvement of both HLA and Ig heavy chain haplotypes in human IgA deficiency.

Immunoglobulin-A deficiency (IgA-D) is the most common human Ig class deficiency with an estimated frequency of approximately 1 in 500 in the Swedish population. We investigated the immunoglobulin heavy chain constant region gene segments (IGHC) in 103 individuals with IgA-D and the immunoglobulin heavy chain variable region gene segments (IGHV) in 20 of these, in order to identify a possible molecular basis of the defect. No deletions of IGHV gene segments of the VH2, VH5, and VH6 families or the IGHG genes were observed. In the IGHC, there were, however, differences in the restriction fragment length polymorphism frequencies of IGHG genes where the Bam HI haplotype "H2" [IGHGP, 10 kilobases (kb), IGHG2, 25 kb; and IGHG4, 9.0 kb] was overrepresented. The mean serum levels of IgG4 and IgE were significantly lower in individuals (both IgA-D subjects and healthy controls) homozygous for the H2 haplotype than in individuals homozygous for the H1 haplotype (IGHGP, 8.8 kb, IGHG2, 13.5 kb, and IGHG4, 9.4 kb). IgA-D subjects homozygous for HLA DQB1*0201 (DQw2), a marker that has previously been reported to show a strong association with IgA deficiency, showed a similar reduction of serum levels of IgG4 and IgE as compared with DQB1*0201 negative IgA-D subjects. These findings suggest that the two loci found to be associated with IgA deficiency may act via a common pathway.


Pharmacokinetics of two oral liquid formulations of oxatomide in children.

A study on the pharmacokinetics of two suspensions of oxatomide at the concentrations of 25 and 2.5 mg/ml was carried out on 12 children (7M, 5F) with a mean age of 9 years and weight of 29 kg. The plasmatic concentration peak, the time to reach the peak and the area under the curve displayed no significant differences between the two formulations. The maximum concentrations and the peak time of oxatomide in children are similar to those observed in adults.


Gastric, intestinal and colonic absorption of a series of beta-blockers in the rat.

Gastric, intestinal and colonic absorption rates of a series of eleven beta-blockers (alprenolol hydrochloride, atenolol, bunolol hydrochloride, penbutolol sulphate, pronethalol hydrochloride, metoprolol, oxprenolol, bevantolol, bufuralol, propranolol hydrochloride and timolol maleate) were estimated using Doluisio's method. The gastric absorption rate was very low and the absorption rate constant could not be assessed accurately in all cases. In the small intestine, the absorption rate constants, Ka, at pH 6.2 ranged between 0.38 h-1 for atenolol and 4.28 h-1 for penbutolol. In the colon, the rate of drug absorption at pH 7.5 ranged between 0.12 h-1 for atenolol and 2.15 h-1 for penbutolol. In most cases, colonic absorption rate constants were of the same order as those obtained in the small intestine, demonstrating the good penetrability through colonic membrane of the series studied. The relationship between absorption rate constants found in the small intestine and colon and the partition constant ([1/Rf]-1), was studied for this non-homologous series of beta-blocker drugs. In both cases, the functional hyperbolic absorption model proposed by Wagner and Sedman [1973] was the most representative.


Antiglucocorticoid action of dehydroepiandrosterone in young obese Zucker rats.

Dehydroepiandrosterone (DHEA) reduces weight gain in the hypercorticosteronemic Zucker fatty rat, an animal model of genetic obesity. However, the mechanism of action of DHEA is still unclear. We propose that DHEA acts as an antiglucocorticoid in the Zucker fatty rat. To test this hypothesis we examined DHEA's ability to block the activation of the glucocorticoid-inducible enzymes tyrosine aminotransferase (TAT) and ornithine decarboxylase (ODC) by dexamethasone (i.p. 5 micrograms/100 g body weight) in hepatic tissue of 6-10 week old Zucker rats. Injections of DMSO, the vehicle, served as a control. DHEA alone did not affect TAT, but when DHEA (500 micrograms/100 g b.w.) was administered simultaneously with dexamethasone, activation did not occur. Similar results were seen using a second tissue (kidney). We conclude that DHEA can act acutely as an antiglucocorticoid in the young obese Zucker rat and hypothesize that its chronic anti-obesity effect may reflect, at least in part, a chronic antiglucocorticoid activity.


Pupillometry in clinical psychophysiological diagnostics: methodology and proposals for application in psychiatry.

The change in pupillary reactions may be considered as an important parameter of the autonomic nervous system. By means of pupillometry, it became possible to measure both objectively and economically these reactions. The method of microprocessor-assisted "static" and light evoked "dynamic" pupillometry is demonstrated. A high reliability for the static variable of pupillometry was also evaluated. Variables measured during "static and dynamic" pupillometry were factor-analyzed. Four factors (static, dynamic, stimulus specific and restitution dependent) were obtained regardless of whether investigations were carried out in normals or in psychiatric patients. Examples of utilization of pupillometry in psychopharmacology and mental disorders are also described in this essay.


[Surgery of chronic pancreatitis].

Surgery for chronic pancreatitis may be indicated for local complications, or if the differential diagnosis between cancer and pancreatitis is uncertain, or if pain does not respond to conservative treatment. Local complications of chronic pancreatitis are the most frequent indications for operation. Pseudocysts are often associated with other local complications, and a high mortality rate is observed when haemorrhage occurs. Duodenopancreatectomy can be performed with low mortality, and is indicated if malignancy cannot be excluded, or in the patient with medically intractable pain in whom a pancreatico-jejunostomy is technically not feasible.


Cellular and enzymatic activities of a synthetic heteropolymer double-stranded RNA of defined size.

We have synthesized a novel heteropolymer double-stranded RNA (dsRNA) molecule of defined length and strandedness (dsRNA309) and evaluated its ability to induce cytokine gene expression, activate dsRNA-dependent enzymes, and inhibit both tumor cell growth and virus replication. Unlike the conventionally studied synthetic homopolymer dsRNAs, polyinosinic acid:polycytidylic acid (poly(I-C)) and its mismatched analogue polyinosinic:polycytidylic, uridylic acid (poly(I-C12,U), dsRNA309 possessed restricted biological activity. dsRNA309 was unable to inhibit tumor cell growth or efficiently induce cytokine (i.e. interferon-beta and interleukin-1 alpha) gene expression. However, dsRNA309 was able to inhibit virus replication and activate dsRNA-dependent intracellular enzymes, 2'-5' oligoadenylate synthetase (2'-5' A synthetase) and the dsRNA-activated inhibitor kinase in in vitro assay systems. Overall, dsRNA309 provided a means for examining the mechanisms governing the dsRNA-regulated antiviral and antiproliferative responses, and studies with dsRNA309 demonstrated that the ability of a synthetic dsRNA to activate dsRNA-dependent intracellular enzymes does not necessarily predict the same gene inducing capacity.


Molecular cloning and chromosomal localization of a human gene encoding D-amino-acid oxidase.

Genomic clones covering the entire sequence of the gene encoding human D-amino-acid oxidase (DAO) (EC 1.4.3.3), one of the principal and characteristic flavoenzymes of peroxisomes, were isolated from human placental genomic libraries with the aid of a previously cloned cDNA for human DAO as a probe. Nucleotide sequence analysis revealed that the gene, present as a single copy in the human genome, comprises 11 exons and spans 20 kilobase pairs. The protein sequences containing the catalytically important residues, Tyr-228 and His-307, are coded for by separate exons. Heterologous transcription initiation sites were identified by primer extension analysis, and the sequence of the 5'-flanking region of the DAO gene was found to show some features common to other mammalian genes, such as those of glucocorticoid and the cAMP-responsive element. An additional noteworthy feature is the presence of promoter-like sequences in the first intron of the gene. In addition, two sequences of alternating pyrimidine and purine nucleotides, (CA)20 and (CA)17, are also present in the first intron. Such sequences may play some role in the expression of the DAO gene in human tissues. With the use of genomic DNAs prepared from human and Chinese hamster somatic hybrid cells as templates for the polymerase chain reaction, the gene for DAO was localized to human chromosome 12.


Multiple DNA elements responsible for transcriptional regulation of the ornithine decarboxylase gene by protein kinase A.

Transcription of the ornithine decarboxylase (ODC) gene is rapidly elevated by activation of protein kinase A (PKA). The additive influence of three cis-acting elements is responsible for this regulation in an adrenal carcinoma cell line. Two sites, CRE2 at -48 base pairs (bp) relative to the start of transcription and CRE3 at +95 bp, are identical to the core motif of the cAMP-responsive element (CRE) of the somatostatin gene and are conserved in the mouse, rat, and human ODC genes. Mutation of CRE2 resulted in a substantial decrease in basal promoter activity, as well as a 5-fold decrease in inducibility of the ODC promoter by PKA. CRE3 did not contribute to the basal activity of the ODC promoter, but mutation of this site resulted in a 2-fold decrease in inducibility by PKA. Deletion of a 45-bp sequence (GC-box) located 5' of CRE2, also resulted in a 2-fold decrease in inducibility of the ODC promoter. DNase I protection revealed the presence of protein binding at CRE2, the TATA box, and the GC-box of the ODC promoter. Mutation of CRE2 resulted in loss of protection of this sequence, as well as the 3' extension of the footprint over the TATA box, without affecting interactions at the GC box. Antibodies to the well characterized CRE-binding protein CREB recognized proteins binding to CRE2, suggesting that binding of CREB, or an antigenically related protein, is important for the activity of CRE2. Additionally, recombinant CREB bound to a DNA probe containing the CRE2 sequence.


Co-expression of insulin and somatostatin genes in pancreatic endocrine cells selected for their high level of insulin gene transcription.

RW cells are pancreatic endocrine RIN cells that have been stably transfected with a chimeric gene that places the expression of the dominant selection gpt gene under the control of the insulin gene regulatory sequences. These RW cells were examined for hormone content using immunocytochemistry. This analysis shows that: first, there are cells that are negative for insulin although they were cultured under selective pressure. Second, there is a higher proportion of somatostatin-producing cells than in the parental RIN cells; these somatostatin cells form two populations: one of cells containing only somatostatin and, surprisingly, one made of cells containing both insulin and somatostatin. Thus: (1) expression of the transfected and endogenous insulin regulatory sequences is not regulated in a coordinate fashion; (2) the presence of both hormones in the same cell suggests that the regulation of the expression of insulin and somatostatin genes and the differentiation pathway of the two respective cell types may be closely related.


Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure.

We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions. The selection is based on the lethality of hydrogen peroxide (H2O2) that is produced in wild type cells during the peroxisomal beta-oxidation of fatty acids. In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes. The mutants were shown to have monogenetic defects and to fall into 12 complementation groups. Representative members of each complementation group were morphologically examined by immunocytochemistry using EM. In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase. These mutants suggest the existence of multiple components involved in peroxisomal protein import. Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation. In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisomes.


Abdominal aortic dissection due to idiopathic medial aortopathy in a 32-year-old Caucasian man.

A case of dissection of the abdominal aorta in a 32-year-old Caucasian man associated with a histological diagnosis of granulomatous aortitis and a clinical diagnosis of idiopathic medial aortopathy is described. The relationship between giant cell "temporal" arteritis, Takayasu's disease and idiopathic medial aortopathy is discussed.


Combined use of beta-adrenergic blocking agents and long-term cardiac pacing for patients with the long QT syndrome.

The objective of this study was to review our current experience using a combination of beta-adrenergic blocking agents and long-term cardiac pacing to treat patients with the idiopathic long QT syndrome. Patients with the idiopathic long QT syndrome are at high risk for sudden cardiac death. Before combination therapy, 20 of the 21 study patients experienced either cardiac arrest (n = 8) or syncope (n = 18) and 11 had documented polymorphous ventricular tachycardia. Nine of these patients had not responded to isolated beta-blocker therapy and five had not responded to isolated left cervicothoracic sympathectomy. All patients were treated with combined beta-blocker therapy and long-term cardiac pacing at a rate designed to normalize the QT interval. Cardiac pacing at rates of 70 to 125 beats/min resulted in shortening of the QT and corrected QT (QTc) intervals from 517 +/- 78 and 541 +/- 62 ms to 404 +/- 37 and 479 +/- 41 ms, respectively. The mean follow-up interval after institution of pacing was 55 +/- 45 months. The only sudden death occurred in a patient who had discontinued beta-blocker therapy. Syncope occurred in four patients, two of whom had interrupted pacemaker function due to lead fracture. Pacemaker problems, partly attributable to the specific rate required for QT interval shortening and to avoidance of T wave sensing, were relatively common. No patient who continued the combination therapy died, but 10% of these patients had a recurrence of symptoms. Combination therapy with a beta-blocker and cardiac pacing appears to be a highly effective primary therapy for symptomatic patients with the long QT syndrome and to provide excellent adjunctive therapy for patients who require insertion of an automatic internal defibrillator.


Preserved ventricular pump function after a marked reduction of left ventricular mass.

This study was designed to evaluate the long-term effects of combination therapy with an angiotensin-converting enzyme inhibitor and a beta-adrenergic blocking agent on the relation between the decrease in arterial pressure at rest and during exercise and the decrease in left ventricular mass. A variety of antihypertensive drugs including angiotensin-converting enzyme inhibitors and beta-blockers have been shown to reduce ventricular hypertrophy, although little is known about combination therapy and the time course of such a reduction. Twenty-one patients with previously untreated essential hypertension were treated with a low dose combination of 50 mg of atenolol and 10 mg of enalapril once daily for 39 months. Cardiovascular findings were assessed by two-dimensionally guided M-mode echocardiography in the pretreatment phase and after 6 and 39 months of combination therapy. Combination therapy reduced arterial pressure at rest from 161/108 to 130/86 mm Hg (p less than 0.001) and exercise arterial pressure at 100 W from 192/112 to 167/95 mm Hg (p less than 0.001). After 6 months of treatment, significant decreases in interventricular septal thickness (9%, p less than 0.001), posterior wall thickness (9%, p less than 0.001) and left ventricular mass index (16%, p less than 0.001) were demonstrated on the echocardiogram. After 39 months of therapy, reductions in these values were 28% (p less than 0.001), 29% (p less than 0.001) and 40% (p less than 0.001), respectively. Long-term treatment with combination therapy of atenolol and enalapril produced significant reductions in arterial pressure at rest and during exercise accompanied by a marked reduction of left ventricular mass. However, whereas arterial pressure decreased immediately and remained unchanged, left ventricular mass decreased more gradually and continued to decrease throughout the treatment period of greater than 3 years. Despite this marked reduction in left ventricular mass, left ventricular pump function was well preserved during rest and exercise.


Effects of budesonide and bambuterol on circadian variation of airway responsiveness and nocturnal symptoms of asthma.

Effects of the inhaled corticosteroid budesonide and the oral beta-agonist bambuterol on the nocturnal worsening of asthma were studied in patients with allergic asthma with a circadian peak expiratory flow variation greater than or equal to 15% (group 1, n = 8) and less than 15% (group 2, n = 9). Airflow limitation and airway responsiveness to histamine were measured during 24 hours after 4 weeks of treatment with (A) 0.4 mg budesonide at 8 AM and 8 PM, (B) 20 mg bambuterol at 8 PM, and (C) placebo, in a randomized, crossover design. Patients in group 1 had worse nocturnal symptom scores and a greater airway responsiveness than patients in group 2; in addition, patients in group 1 had a larger increase in responsiveness during the night (1.1 versus 0.6 doubling concentrations [DC]). Both budesonide and bambuterol improved responsiveness more at night than during daytime, thus decreasing circadian variation: at 4 AM, increases in PC20 were 2.0 DC after budesonide and 0.8 DC after bambuterol, compared with placebo. Bambuterol reduced circadian variation in FEV1, but in contrast to budesonide, did not improve 24-hour mean levels of FEV1 and PC20. Both drugs beneficially influenced nocturnal symptoms; the effects of budesonide were stronger than those of bambuterol. Our findings demonstrate that budesonide and bambuterol reduce nocturnal airway responsiveness and asthma symptoms and suggest a relationship between the degree of airway responsiveness and the presence of nocturnal symptoms of asthma.


From Vineberg to bypass: a "second-hand" internal mammary artery.

A patient operated upon 20 years ago for myocardial revascularization with two Vineberg procedures and one coronary artery bypass graft (CABG) on the right coronary artery had a recurrence of unstable angina due to the thrombosis of the left internal mammary artery (IMA) and the right CABG. The right IMA was patent but stenosed distally. Reoperation was performed with a direct end to side anastomosis of the patent right IMA onto the left anterior descending artery and a saphenous vein graft as a CABG on the marginal branch. We comment on the choice of this "second hand" IMA graft and the pathological appearances of the patent IMA.


Synaptic transmission in rat cardiac neurones.

Intracellular recordings of spontaneous synaptic activity and synaptic responses to fibre tract stimulation were taken from neurones of ganglia isolated from the left atrium and interatrial septum of the rat. In six out of 57 neurones studied, spontaneous fast excitatory postsynaptic potentials (EPSPs) were recorded. Single stimulation of fibre tracts approaching the ganglion resulted in an all-or-none response consisting of an EPSP, from which an action potential abruptly appeared. This response disappeared in Ca(2+)-free/high-Mg2+ solution, indicating that it was orthodromic in origin. EPSPs were markedly exaggerated and prolonged by neostigmine (1-5 microM). EPSPs produced by high-frequency (0.1-20 Hz) fibre tract stimulation were markedly attenuated when compared with responses to single fibre tract stimulation, although they usually remained suprathreshold for spike initiation. High concentrations of hexamethonium (1 mM) and d-tubocurarine (300 microM) failed to inhibit responses to single fibre tract stimulation, although they completely abolished responses to high-frequency stimulation. Responses to single fibre tract stimulation were abolished by trimetaphan (greater than or equal to 100 microM). No slow synaptic responses were detected during single or high-frequency fibre tract stimulation. All cardiac neurones that responded orthodromically were highly excitable: they had a short post-spike after-hyperpolarization (AHP) and responded with multiple firing to prolonged membrane depolarization. It is concluded that cardiac neurones, in the region of the heart studied here, receive single 'strong' cholinergic inputs from some fibre tracts approaching the ganglion that elicit EPSPs accompanied by spikes. EPSPs are rather resistant to ganglion-blocking agents and subject to frequency modulation.


Effects of consecutive administration of central and peripheral anticholinergic agents on respiratory sinus arrhythmia in normal subjects.

Respiratory sinus arrhythmia is thought to be vagally mediated, since it disappears after atropine, but the site of action of the drug (central vs. peripheral) accounting for this effect has not been elucidated. To investigate the effects of anticholinergic agents on respiratory arrhythmia, ten healthy subjects received an intravenous bolus of tropatepine (a presumed central antagonist) at a dose of 0.08 mg per kg of body weight, then, 7 min later, prifinium (a peripheral antagonist) at a dose of 0.1 mg per kg of body weight. Respiratory sinus arrhythmia during controlled breathing was evaluated as the area under the high-frequency peak of the heart rate variability spectrum coinciding with the respiratory frequency +/- 0.02 Hz. The power of this high-frequency peak decreased by 55% after tropatepine (P less than 0.05) with a concomitant increase of the mean RR interval from 930 to 1072 ms (P less than 0.01). When prifinium was added, a further but non-significant decrease of respiratory arrhythmia was observed, while the mean RR interval decreased from 1072 to 714 ms (P less than 0.01). The low-frequency components (0.05 to 0.15 Hz) of the power spectrum, significantly decreased (P less than 0.05) after infusion of both drugs. In conclusion, tropatepine depresses respiratory sinus arrhythmia with a paradoxical concomitant bradycardia. This suggests that tropatepine acts like a pure central muscarinic antagonist, in support of the hypothesis that a central cholinergic receptor is involved in the respiratory modulation of heart rate.


Amino acid gradients across the intestinal circulation in fetal lambs.

Amino acids, including glutamine, glutamate and asparagine are major metabolic substrates for the adult enterocyte of several species. To determine whether circulating amino acids are utilized by the fetal intestine, we studied nine fetal sheep (mean gestational age 128 +/- 5 days; term: 147 days). Catheters were inserted into the descending aorta (DA) and the mesenteric vein (MV) to allow for simultaneous blood sampling across the intestine. Fetal blood gas, haemoglobin; O2 saturation and O2 tension were measured. Ammonia was determined by an enzymatic method and HPLC analysis was used to measure the content of all amino acids in DA (descending aorta) and MV (mesenteric vein). Intestinal blood flow measurements were obtained using the radionuclide microsphere method. Intestinal blood flow (81 +/- 28 ml/min/100g) and arterial pH (7.37 +/- 0.04) were within normal range for unstressed fetal lambs. Glutamine and glutamate were the only amino acids that were significantly taken up across the fetal intestinal circulation. The fetal intestine extracted approximately 21% of the delivered glutamine (6.8 +/- 4.5 mumol/min/100g), 7% of the delivered glutamate (1.3 +/- 1.1 mumol/min/100g) and 2.7% of the delivered oxygen (43.0 +/- 19.1 mumol/min/100g). These data suggest that glutamine and glutamate are major substrates for the intestine in unstressed fetal lambs.


Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.


Prolonged IL-2 receptor alpha/CD25 expression after T cell activation via the adhesion molecules CD2 and CD28. Demonstration of combined transcriptional and post-transcriptional regulation.

The T cell adhesion molecule CD28 provides a costimulatory signal in combination with CD2 and CD3 mAb. CD28 regulates the expression of cytokines by T cells, not only IL-2, but also IL-1 alpha and CSF-1, usually synthesized by accessory cells. We have investigated the mechanisms through which CD28 modulates the expression of the IL-2R alpha chain. Whereas activation through CD2 or CD28 alone induced no or only low IL-2R alpha chain expression, activation through CD2 plus CD28 led to both a high and prolonged (greater than 14 days) cell surface and mRNA expression. In contrast, immobilized CD3 mAb-dependent activation induced a transient expression of the IL-2R alpha chain, which was neither further increased nor prolonged by CD28 costimulation. Upon CD2 plus CD28 stimulation, the half-lives of the two IL-2R alpha transcripts increased progressively between days 1 and 4, in contrast to each pathway alone. Whereas each activation pathway alone induced either no (CD2) or low (CD28) levels of IL-2R alpha gene transcription, the CD2 plus CD28 stimulation was associated with its increased transcription, which persisted at similar rates between 5 and 96 h post stimulation. The in vitro costimulation via the CD2 and CD28 molecules thus regulates the expression of the IL-2R alpha gene both at the transcriptional and post transcriptional levels. Our results therefore demonstrate a new immunoregulatory function of the CD28 molecule on IL-2R alpha expression, which, through its increased transcription rate and stabilization, could, together with high levels of cytokines secretion, be responsible for the prolonged T cell proliferation.


Regulation of LFA-1 avidity in human B cells. Requirements for dephosphorylation events for high avidity ICAM-1 binding.

Regulation of the avidity of LFA-1 (CD11a/CD18, alpha L beta 2) for its ligand ICAM-1 (CD54) was studied in human B cells by evaluating the effects of a phorbol ester, anti-IgM antibodies, staurosporine, and okadaic acid. We monitored changes in LFA-1 avidity by quantifying binding of cells to an immobilized rICAM-1 fusion protein. In this assay, the protein kinase C-activating phorbol ester PDB and anti-IgM antibodies, as well as the protein kinase inhibitor, staurosporine, were able to induce LFA-1-dependent binding to ICAM-1. This demonstrates that the high avidity state of LFA-1 can be induced by a protein kinase C-dependent and by a protein kinase C-independent pathway. Furthermore, treatment of the cells with the protein phosphatase inhibitor, okadaic acid, inhibited binding to ICAM-1. Treatment with staurosporine before addition of okadaic acid not only induced enhanced binding of cells to ICAM-1, but also dramatically reduced the ability of okadaic acid to inhibit binding. These results suggest a critical role for a protein phosphatase in inducing the high avidity state of LFA-1 as well as a role for a protein kinase in inducing the low avidity state of LFA-1.


IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.


Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages.

Taxol, a microtubule stabilizing agent, exhibits promise in the treatment of breast and ovarian tumors. Recently, this novel drug has been shown to activate murine macrophages to express TNF-alpha and to down-regulate TNF-alpha receptors, activities shared by bacterial LPS. Our study sought to determine if taxol could regulate gene expression in murine macrophages and to examine further the ability of taxol to generate an LPS-like signal. Toward this end, the ability of taxol to induce TNF-alpha mRNA and five other genes (IL-1 beta, IP-10, D3, D7, and D8) associated with LPS-activation of macrophages was examined by Northern blot analysis. Taxol alone (1-30 microM) induced murine C3H/OuJ macrophages to secrete bioactive TNF-alpha and express increased levels of each of the six genes under investigation. The magnitude and the kinetics of induction of each gene closely resembled that seen with Escherichia coli K235 LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, however, failed to induce detectably any of the genes in response to taxol, despite being sensitive to the microtubule stabilizing effects of taxol as determined by immunofluorescence microscopy. The gene induction activity of taxol was in marked contrast to an alternative macrophage activator, heat killed Staphylococcus aureus, which induced a distinct gene profile in C3H/OuJ macrophages and which was equally active in C3H/OuJ and C3H/HeJ macrophages. These data are consistent with an ability of taxol to generate an LPS-like signal, possibly through a common signaling intermediate. As a first step toward identifying signal responses shared by taxol and LPS, we have shown that taxol, as shown previously for LPS, rapidly induces the tyrosine phosphorylation of a 41- and 42-kDa protein.


Detection of human immunodeficiency virus type 2 in brain tissue.

Infection due to human immunodeficiency virus (HIV) type 2 is believed to cause a clinical picture similar to that of HIV-1, although extensive data are not available. In 2 patients with West African exposure and neurologic symptoms, HIV-2 was detected in the central nervous system using DNA and RNA polymerase chain reaction, in situ hybridization, and immunohistology. In the first patient, the neurologic disease was most likely due to productive infection with HIV-2. In the second, a combination of neuropathologic abnormalities (including the presence of HIV-2) explained the clinical features. Thus HIV-2, like HIV-1, can be readily detected in brain tissue in patients with neurologic abnormalities, although the exact role of HIV-2 in pathogenesis of AIDS-associated neurologic disease requires further study.


Intramolecular transformation reaction of the glutathione thiyl radical into a non-sulphur-centred radical: a pulse-radiolysis and EPR study.

The thiyl radical derived from glutathione (GSH) is shown to decay rapidly in aqueous solution by intramolecular rearrangement reactions into the non-sulphur-centred radical 1. The reaction is induced by OH- with a rate constant of 5 x 10(9) dm3 mol-1 and is also observable at near-neutral conditions (at physiological pH values around 7.5 the rate of formation of 1 amounts to approximately 1 x 10(3) s-1). The activation enthalpy and entropy at pH 8.4 and 20 degrees C were found to be 26.7 kJ mol-1 and -77 J mol-1 K-1, respectively. Radical 1 was unequivocally identified by EPR as the alpha-amino radical at the glutamyl residue of GSH. It is relatively long-lived with typical bimolecular decay rate constants of the order of (2-20) x 10(6) dm3 mol-1 s-1. At higher GSH concentrations the formation of 1 is retarded but not inhibited. All radicals, sulphur- as well as non-sulphur-centred ones are connected via equilibria, partly under the action of 'repair' processes of GSH. These repair processes, however, are slow (k much less than 1.4 x 10(5) dm3 mol-1 s-1). The equilibria are established quite rapidly and were found to be far on the side of the non-sulphur-centred radical under all conditions employed. Radical 1 possesses reducing properties as evidenced by its fast reaction with 4-nitro-acetophenone (PNAP) to yield PNAP.- (k = 7 x 10(8) dm3 mol-1 s-1).


Heavy ion-induced DNA double-strand breaks in yeast.

DNA double-strand break (dsb) induction in diploid yeast was measured by neutral sucrose sedimentation after exposure to very heavy ions with values of linear energy transfer (LET) ranging from about 300 to 11500 ke V/microns. Linear fluence dependencies were found in all cases from which dsb production cross-sections (sigma dsb) could be calculated. Corresponding cross-sections for cell killing (sigma i) were derived from final slopes of survival curves measured in parallel and for the same fluence range. A close correlation was found between sigma i and sigma dsb. It is calculated that over the entire LET range, including 30 MeV electron irradiation, about 22 dsb are induced per lethal event when high exposures are considered.


The role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s-1 to 144 s-1, and for lethal lesion precursors from 533 s-1 to 165 s-1. This is generally consistent with the role of GSH in the repair-fixation model of radiation damage at the critical DNA lesions. However, the reduction in chemical repair rate was not proportional to the severe thiol depletion (down to approximately 1% for GSH) and a residual repair capacity remained (approximately 30%). This was found not to be due to compartmentalization of residual GSH in the nucleus, as the repair rate for dsb precursors in isolated nuclei, washed virtually free of GSH, was identical to that found in GSH-depleted cells (144 s-1), also the OER remained substantially above unity. This suggests that other reducing agents may have a role to play in the chemical repair of oxygen-dependent damage. One possible candidate is the significant level of protein sulphydryls present in isolated nuclei.


The influence of ras oncogene expression on radiation response in the Rat-1 cell.

We evaluated the effect of H-ras oncogene expression on resistance to ionizing radiation in cultured rat fibroblasts. The Rat-1 cell line, and two Rat-1 derivatives, MR4 and MR7, carrying a ZN-regulatable metallothionein-rasT24 fusion gene were used to study the effects of the ras oncogene on radiation sensitivity. Cells were irradiated with a 137Cs source (450 cGY/min) in the presence or absence of ZnSO4. Multiple cell survival studies did not show an appreciable difference in sensitivity to radiation among the lines in the presence or absence of ras oncogene expression.


Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells.

DNA breaks in eukaryotic cells can be detected by alkaline electrophoresis of cells embedded in agarose. DNA containing breaks extends in the direction of the anode forming an image resembling the tail of a comet. We have adapted this procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m-2 and incubation for as little as 5 min. We have also used SCGE to study UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, we have incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examining the consequent breaks both by SCGE and by alkaline unwinding in order to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10(9) daltons are detected.


Synergism between electrolysis and methylene blue photodynamic action in Escherichia coli.

There is interest in the use of photodynamic therapy for the treatment of certain diseases, including cancer. However, weak penetration of visible light in tissues has restricted its use. In this study the possibility of enhancing photodynamic effects by the use of energies that penetrate more deeply in tissues was investigated. Weak electric currents (1.0 mA) applied to Escherichia coli cells for short periods, producing little or no lethal damage, was found to act synergistically with the photodynamic action of methylene blue, significantly enhancing the effects of this treatment. This synergism exists also between electrolysis and X-rays but not between electrolysis and UV-254 nm. It is suggested that this synergism might eventually be used to improve the results obtained in therapeutic practice based on the utilization of photodynamic action.


EPR study of mouse tissues in search for adaptive responses to low level whole-body X-irradiation.

Effect of a low dose of whole-body X-irradiation (D1 = 0.075 Gy) of mice on some biochemical changes induced in the spleen and thymus by a subsequent challenge dose (D2 = 1.5 Gy) was studied by electron paramagnetic resonance (EPR). Kunming and SHK mice were used. Concentration of ribonucleotide reductase (RR) in the spleen and thymus of unirradiated Kunming mice was 0.70 and 0.46 microM respectively, and of unirradiated SKH mice--0.37 and 0.21 microM respectively (systematic error 45%). For mice exposed only to the low dose (D1 group), a stimulating effect on RR activity in spleen and thymus was found, while in mice subjected only to the D2 dose (D2 group) activity of the enzyme in the organs decreased considerably 18 h after irradiation. The group of mice irradiated with D1 and D2 doses, given at a 6-h interval (D1 plus D2 group), showed a RR activity in the organs lower than D1 and higher than D2 groups. This finding indicates that there exists an adaptation-like response to a low-dose whole-body irradiation in murine spleen and thymus. This low 'inductive' dose makes the organs' RR less susceptible to inhibition induced by a subsequent challenge dose. Preliminary results showed that the same kind of response is probably a characteristic of the RR content of splenocytes as well as of the rate of thymidine incorporation into splenocytes. 5-Thyml radicals of DNA (TH radicals) induced in whole tissues by gamma-irradiation in vitro at 77 K were also studied. Radiochemical yield of these radicals (4.0 and 5.3 nmol j-1), for spleen and thymus respectively of unirradiated Kunming mice, and 2.3 and 2.6 nmol J-1, for spleen and thymus respectively of unirradiated SHK mice (systematic error 30%), decreased significantly in both organs upon D2 irradiation. This decrease, however, was the consequence of DNA content diminishing upon D2 irradiation rather than change of DNA radiosensitivity: the beta value of TH radicals, i.e. yield of radicals per unit mass of DNA in each organ was equal for the mice from all D1, D1 plus D2, D2 and control groups. The beta values of TH radicals in mouse spleen and thymus were of the same order of magnitude compared with the yield of the single-strand breaks of DNA measured previously in rat organs just after whole-body irradiation, i.e. about 1 x 10(2) (Gy x 10(12) Da)-1.