gamma-Aminobutyric acid-opioid interactions in the regulation of gonadotropin secretion in the immature female rat. Previous studies in the male rat have demonstrated that GABA acting via GABAB receptors can abolish naloxone-induced LH secretion. The purpose of the present study was to determine if an analogous situation exists in the female rat. Naloxone administered to ovariectomized immature rats had no effect on LH release. In contrast, naloxone potently stimulated LH release in estrogen-primed ovariectomized immature rats. Elevation of endogenous brain levels of GABA by administering amino-oxyacetic acid, an inhibitor of GABA catabolism, prevented the naloxone-stimulated release of LH. This effect appeared to be both GABAA and GABAB receptor mediated, since exogenous administration of either muscimol (GABAA agonist) or baclofen (GABAB agonist) prevented the naloxone-induced release of LH. Neither GABA agonist had any effect on LHRH-stimulated LH release in vivo, suggesting that their effect was specific and achieved at the level of the CNS. In contrast to its inhibitory effect on naloxone-stimulated LH and FSH release, muscimol increased basal LH and FSH release in vivo and from hemipituitaries incubated in vitro, while having no effect on LHRH release from mediobasal hypothalamic and preoptic area fragments in vitro. Thus, under conditions of basal LH release, activation of GABAA receptors in the anterior pituitary can actually lead to enhanced LH secretion. Finally, naloxone-stimulated LH release was found to be inhibited by the alpha 1- and alpha 2-adrenergic blockers, prazosin and yohimbine, suggesting that naloxone-stimulated LH release is mediated via catecholamine neurotransmission involving alpha 1- and alpha 2-adrenergic receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)


In vitro regulation of pituitary ACTH secretion in inflammatory disease susceptible Lewis (LEW/N) and inflammatory disease resistant Fischer (F344/N) rats. We have previously shown that susceptibility to inflammatory disease in Lewis (LEW/N) rats is related to their limited hypothalamic-pituitary-adrenal (HPA) axis responses to a variety of inflammatory stimuli, while the relative resistance to inflammatory disease in Fischer (F344/N) rats is related to their potent HPA axis responses to these same stimuli. In vivo studies also showed that LEW/N pituitary ACTH responses to exogenous corticotropin-releasing hormone (CRH) were blunted compared to F344/N. To determine if there is a fundamental difference in pituitary corticotroph function between the two strains, independent of other factors influencing the HPA axis, we compared ACTH responses to a variety of stimuli in LEW/N and F344/N primary pituitary cell cultures. Here we show that in vitro basal ACTH secretion and peak ACTH response to CRH, forskolin and 8-bromo-cAMP are 50% lower in LEW/N than F344/N rats. However, these findings can be explained by other observations: diminished basal ACTH content, POMC mRNA, and a decreased number of corticotrophs, in pituitary cell cultures from LEW/N compared to F344/N rats. In addition, LEW/N corticotrophs were more sensitive to dexamethasone and to corticosterone suppression of CRH-stimulated ACTH secretion compared to F344/N. The data support the possibility of an HPA axis defect in LEW/N rats at the pituitary level which could be secondary to prolonged understimulation by hypothalamic CRH, or could also be partially related to enhanced glucocorticoid feedback inhibition.


Localization and quantification of pro-opiomelanocortin mRNA and glucocorticoid receptor mRNA in pituitaries of suicide victims. Suicidal behavior has been associated with hypothalamic-pituitary-adrenal overactivity in humans, as measured by increased corticosteroid secretion. To investigate whether this overactivity is reflected at the pituitary level, we have studied the localization of pro-opiomelanocortin (POMC) mRNA, and glucocorticoid receptor (GR) mRNA, in human anterior pituitaries, and quantified these messages relative to controls. Pituitaries from 7 suicide victims and 11 cardiac deaths were sectioned into 10-microns slides, stained with thionin and processed for in situ hybridization using a riboprobe complementary to human POMC mRNA. To correct for possible postmortem cell loss, hybridization with P1B15, a cDNA complementary to rat cyclophillin mRNA, was used in adjacent sections. POMC mRNA containing cells were found to be localized in clusters and were highly associated with corticotropin-releasing hormone (CRH) receptors. In contrast, GR mRNA containing cells were distributed through the pituitary, although areas of increased density were associated with POMC mRNA cells. Quantification with a computerized image analysis system revealed a 25% increase in POMC message in suicide victims. Analysis of the corticotrophic cell clumps showed that the suicide victims had higher POMC mRNA density per cell (p = 0.04) and larger corticotrophic cell size (p = 0.04) than the cardiac death victims. No differences in GR mRNA were detected between the two groups, although GR and POMC mRNA levels were highly and significantly correlated (r = 0.8, p < 0.001). There were no differences in P1B15 message between the two groups. We conclude that in situ hybridization is a useful tool to study gene regulation in human neuroendocrine tissue and that suicide victims show evidence of chronic hypothalamic-pituitary-adrenal axis activation.


A central mechanism is involved in the secretion of ACTH in response to IL-6 in rats: comparison to and interaction with IL-1 beta. The cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha are known to be potent effectors of ACTH secretion. Some of the peripheral effects of IL-1 beta appear to be related to the secretion of IL-6 induced by IL-1 beta. Thus, we evaluated the effect of IL-6 on ACTH secretion and its interaction with IL-1 beta. Rats received recombinant human (rhIL-6) or murine (rmIL-6) IL-6 through indwelling jugular cannulae. rhIL-6 (200 ng or 2 micrograms/rat) produced peak plasma ACTH levels which were 3- to 4-fold greater than basal levels. rmIL-6 produced similar responses. Neither species of IL-6 affected plasma prolactin levels. Comparison of rhIL-1 beta (200 ng) to rhIL-6 (200, 100 or 50 ng) showed that IL-6 elevated ACTH in a dose-dependent manner and that IL-1 beta was significantly more effective. IL-1 beta was also administered concomitantly with or 10 min after IL-6. Delivered together, IL-1 beta (100, 30 or 10 ng) and IL-6 (100 ng) produced significantly higher ACTH levels than when given alone. This additivity was also evident when IL-6 was given 10 min prior to IL-1 beta. The coadministration of IL-6 (2 micrograms) with corticotropin-releasing factor (CRF, 1 micrograms/kg, b.w.) also had an additive effect on ACTH secretion (at 20 min: 300 +/- 40 pg/ml for CRF; 320 +/- 83 pg/ml for IL-6; and 540 +/- 44 pg/ml for CRF + IL-6), whereas a higher dose of CRF (10 micrograms/kg b.w.) yielded ACTH levels of 1,000 +/- 107 pg/ml at 20 min, with no further enhancement by IL-6. Incubation of pituitary cells with IL-6 alone (0.1, 1.0 or 3.0 nM) produced a slight but significant stimulation of ACTH secretion within 2 h in response to the higher doses of IL-6 only (p < 0.05), but did not modify the effect of CRF in vitro. To determine if the action of IL-6 was at a site(s) within the brain, IL-6 (30 or 100 ng/0.5 microliters) was injected into the third cerebroventricle of alert rats. 100 ng IL-6 elicited peak plasma ACTH levels (300 +/- 65 pg/ml) within 30 min; these were significantly higher than the buffer responses (90 +/- 25 pg/ml, p < 0.01), and lower than the responses to 30 ng IL-1 beta (530 +/- 50 pg/ml, p < 0.001). 30 ng IL-6 was ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)


Treatment of human cutaneous sporotrichosis with itraconazole. Eighteen adult white male patients with cutaneous sporotrichosis were treated with itraconazole following different daily dose schemes. Cure was obtained in all cases after periods of 15-75 days (median 44 days) with total doses between 3.1 and 14.8 g (median 8.4 g). No serious side effects were observed and no relapses occurred in the follow-up period of between 1 and 26 months (median 14.7). These results show that itraconazole represents a safe and effective drug for the treatment of sporotrichosis. Comparison with other studies leads us to consider a daily dose of 200 mg as the most appropriate. A concomitant warming of the affected limbs should be recommended.


Effect of metyrapone on the pituitary-adrenal axis in depression: relation to dexamethasone suppressor status. It has been suggested that the well-documented hypercortisolaemia found in a proportion of patients with severe depression occurs either in response to excessive secretion of corticotrophin-releasing hormone-41 (CRH-41) from the hypothalamus, or as a consequence of up-regulation of pituitary CRH-41 receptors. The attenuation of the normal ACTH response to CRH-41 in these subjects is thought to result from inhibition of corticotrophin secretion by elevated cortisol levels. We tested these hypotheses by examining ACTH responses to metyrapone, an 11 beta-hydroxylase inhibitor which blocks the formation of cortisol, followed by CRH-41 in 15 severely depressed in-patients diagnosed according to DSM-IIIR criteria. Patients were assigned to two groups according to their response to overnight administration of 1 mg dexamethasone: suppressors (8) and nonsuppressors (7). A third group consisted of 6 healthy matched controls. Metyrapone 750 mg was given 4-hourly for 24 h and samples were taken for cortisol and ACTH. Six of the original 15 patients (3 from each group) were given a bolus dose of 100 micrograms human CRH-41 intravenously after 24 h of metyrapone, and ACTH levels were measured over 2 h. Falls in circulating cortisol in response to metyrapone were similar in all three groups. However, we found exaggerated rises in ACTH amongst the nonsuppressors, as compared to the suppressors and the control group, after metyrapone.(ABSTRACT TRUNCATED AT 250 WORDS)


The modulation of dopaminergic transmission in the striatum by MK-801 is independent of presynaptic mechanisms. This paper reports biochemical and behavioural experiments, planned to obtain a deeper knowledge on the mechanisms of the facilitating action of dopaminergic transmission, induced by the NMDA-sensitive glutamate receptor antagonist, dizocilpine (MK-801). Single or repeated administrations of MK-801 (0.25 mg/kg, i.p., daily for 21 consecutive days) failed to change levels of either dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) in the striatum of the rat or the haloperidol-induced (0.125 mg/kg, i.p.) accumulation of DOPAC. Consistently, the NMDA antagonist, given at a dose which did not affect the spontaneous motility of the animal (0.125 mg/kg, i.p.), failed to potentiate the behavioural stimulatory effect, induced by the dopaminomimetic agents, methamphetamine or nomifensine. All these results, taken together, exclude a facilitating action of MK-801 on dopaminergic neurotransmission. The possibility that the stimulatory effect of MK-801 on dopaminergic neurones is indirect and independent of presynaptic mechanisms is discussed.


Opioid receptors mediating antinociception from beta-endorphin and morphine in the periaqueductal gray. beta-Endorphin and morphine produce an increase in the latency of the tail-flick reflex when administered into the PAG of awake rats. The antinociceptive effect of both opioid agonists was blocked by the sequential local injection of either CTP (D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2), a selective mu opioid receptor antagonist, naltrexone, or beta-endorphin (1-27), a putative epsilon opioid receptor antagonist, with minimal selectivity. When either CTP or naltrexone was used as the antagonist, the dose-inhibition curves generated for beta-endorphin and morphine were not parallel, suggesting the involvement of separate and distinct receptors. Also, synergism occurred when a dose of morphine producing submaximum antinociception was administered simultaneously with either a submaximal or ineffective dose of beta-endorphin. Inhibition of the antinociceptive response to beta-endorphin by mu antagonists and the non-selective antagonism of both beta-endorphin and morphine by beta-endorphin (1-27) suggested that epsilon opioid receptors were not involved. Additionally, a mu/delta opioid receptor complex was not involved, since ICI 174,864 (Allyl2-Tyr-Aib-Aib-Phe-Leu-OH), a selective delta opioid receptor antagonist, did not alter the response to beta-endorphin. Thus, although additional characterization is required, beta-endorphin and morphine appear to act (at least in part) through different opioid receptors, demonstrable using selected mu opioid receptor antagonists.


ACTH-like molecules in lymphocytes. A study in different vertebrate classes. Immunocytochemical and cytofluorimetric studies were performed in several species of different classes (Pisces, Amphibia, Reptilia and Aves), in order to ascertain the presence of ACTH-like molecules in blood cells. Using monoclonal and polyclonal antibodies, we demonstrated that lymphocytes containing ACTH-like molecules are first detectable in anuran amphibia, and are present in lymphocytes of reptilia and aves. Taking into account previous results demonstrating that ACTH-like molecules are present in and modulate macrophage functions from molluscs to man, it is argued that a new biological role has probably been acquired by ACTH in higher vertebrates, where it is also expressed in cells belonging to the lymphocyte lineage, possibly exerting a regulatory role on antibody response.


A fatal infection due to Fusarium oxysporum in a child with Wilms' tumour. Case report and review of the literature. Fusarium oxysporum was isolated twice from the blood culture of a 5-year-old boy with inoperable Wilms' tumour (stage IV) 4 weeks after a cytoreductive therapy with actinomycin D, vincristine and adriamycin. The child died 3 weeks after the first isolation of the fungus with signs of hepatic failure and consumptive coagulopathy. The importance of infection with Fusarium spp. in immunocompromised neutropenic patients and their pathogenetic role are discussed in the view of the literature.


Psychoimmunoendocrine aspects of panic disorder. Immunological, neuroendocrine and psychological parameters were examined in 14 psychophysically healthy subjects and in 17 panic disorder patients before and after a 30-day course of alprazolam therapy. T lymphocyte proliferation in response to the mitogen phytohemagglutinin, lymphocyte beta-endorphin (beta-EP) concentrations, plasma ACTH, cortisol and beta-EP levels were examined in basal conditions and after corticotropin-releasing hormone (CRH) stimulation. Cortisol inhibition by dexamethasone (DST) and basal growth hormone (GH) and prolactin levels were also examined. Depression, state or trait anxiety, anticipatory anxiety, agoraphobia, simple and social phobias, severity and frequency of panic attacks were monitored by rating scales. The immune study did not reveal any significant difference between patients and controls, or any effect of alprazolam therapy. The hormonal data for the two groups were similar, except for higher than normal basal ACTH and GH plasma levels, lower than normal ratios between the ACTH and cortisol responses to CRH, and blunted DST in some patients. All the impairments improved after alprazolam therapy, in parallel with decreases in anxiety and in severity and frequency of panic attacks.


Gamma-aminobutyric acid-B (GABAB) binding sites in postmortem suicide brains. Gamma-aminobutyric acid-B (GABAB) binding sites labelled with [3H]GABA were determined in postmortem frontal cortex samples of 20 control subjects and 16 suicides. The suicide group was further subdivided according to the method of suicide and the existence of depressive symptoms prior to death. No significant differences in GABAB binding were found either between overall suicide and control groups or between the control group and the other subgroups (violent suicide, nonviolent suicide, nondepressed and depressed suicide victims). A significant increase in GABAB binding was observed in those individuals dying from carbon monoxide poisoning. It is concluded that although GABAB binding sites are not altered in our suicide group, a presynaptic dysfunction might account for the increased GABAB binding found in the carbon monoxide subgroup.


Constant light exposure increases 2-[125I]iodomelatonin binding sites in the guinea pig spleen. The effect of constant light exposure on 2-[125I]iodomelatonin binding sites in the guinea pig spleen was investigated. Guinea pigs exposed to constant light for 2-3 weeks showed an increase in the number of splenic 2-[125I]iodomelatonin binding sites, without any difference in the binding affinity, in comparison to animals kept under 12 h light/12 h darkness condition, whereas the splenic indices were similar in both groups. This light dependence of the splenic 2-[125I]iodomelatonin binding sites suggests that the binding sites are melatonin receptors and that the light dependent immunomodulatory effect of melatonin is mediated via a direct action on the lymphoid tissue.


Involvement of opioid receptors in nucleus tractus solitarii in modulating endotoxic hypotension in rats. Opioid antagonists selective for the mu-receptor (naltrexone) and kappa-receptor (nalmefene), respectively, were unilaterally microinjected into the nucleus tractus solitarii (NTS) of the pentobarbital anaesthetized (50 mg/kg, i.p.) rat subjected to E. coli endotoxin (15 mg/kg, i.v.). Naltrexone (0.5 microgram) ameliorated or totally abolished the endotoxic hypotension and tachycardia, and nalmefene at a dose of 1/25 (0.02 microgram) that of naltrexone showed a similar effect. The results suggest that the kappa- and mu-opioid receptors in NTS are actively involved in mediating the hypotensive and tachycardiac effects induced by systemic endotoxin in rats.


An N-ethylmaleimide-sensitive G-protein modulates Aplysia Ca2+ channels. The sulfhydryl alkylating agent N-ethylmaleimide (NEM) was used to probe the possible modulation of calcium current (ICa) by G-proteins in identified neurons of Aplysia californica. ICa recorded with conventional two-electrode voltage clamp was irreversibly suppressed by bath applied NEM in a concentration-dependent manner. This effect was fully blocked by addition of dithiothreitol or intracellular pressure injection of GTP gamma S but was unaffected by pre-treatment with pertussis toxin. These findings suggest that NEM inhibits ICa by causing persistent activation of an inhibitory G-protein in the absence of applied agonist. It appears that alkylation of key cysteine residues involved in G-protein deactivation underlie this effect.


Intracellular pH of tissue-cultured bovine corneal endothelial cells. Intracellular pH (pHi) of bovine tissue-cultured corneal endothelial cells has been measured under several experimental conditions. Determinations were made on individual cells using video-imaging techniques that allowed assessment of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence at 440 and 490 nm. Each experiment had a calibration performed on a cell monolayer: this was performed using a high K(+)-nigericin solution. Resting pHi was 7.25 +/- 0.03 (n = 18) in bicarbonate solution at pH 7.4. Amiloride (1 mM) caused an acidification of approximately 0.2 U within 2 min: replacement with normal Ringer allowed a return to normal pHi after an alkali overshoot. Exposure to 20 mM NH4Cl caused alkalinization that became acidic upon washout of NH4Cl. In Na(+)-rich solution pHi returned to normal after acidification but pHi remained low in Na(+)-free solution until substituted by Na(+)-rich solution. Removal of HCO3- from the bathing solution caused a nonsignificant acidification of pHi by 0.1 U at 2 and 4 min, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) acidified pHi by 0.14 U at 2 min and 0.24 U at 4 min. Addition of DIDS (1 mM) in a HCO3(-)-free solution had no effect on pHi. Hydrogen peroxide acidified pHi by 0.3 U at 50 microM and 1 mM. These results indicate that a Na+:H+ antiport exists that regulates pHi even at normal ambient pH in the presence of bicarbonate: this process becomes highly activated after an acid load. There is a DIDS-sensitive HCO3- movement that is probably coupled to Na+ or Cl-.


Toxic effects of 5-fluorouracil on fibroblasts following trabeculectomy. The inhibitory effect of 5-fluorouracil (5-FU) on fibroblast proliferation is well established. In addition, toxic effects of 5-FU on existing fibroblasts, in rabbits and in vitro, were demonstrated. We examined human subconjunctival scar tissue which was removed during Molteno tube implantation. Surgery was performed 9 weeks after filtering surgery with 5-FU that resulted in bleb scarring. In the tissue, intracytoplasmic vacuoles were detected in some myofibroblasts, with no visible collagen in their vicinity. This presumed toxic effect of 5-FU may be one explanation for the adequacy of fewer than twice 5-FU injections daily following filtering surgery, and for less than 14 days, as originally recommended for inhibiting bleb scarring.


Recombination of nicked DNA knots by gamma delta resolvase suggests a variant model for the mechanism of strand exchange. Fast and efficient recombination catalyzed by gamma delta resolvase in vitro requires negative DNA supercoiling of plasmid substrates. The current model for recombination suggests that supercoiling is required to drive DNA strand exchange within a synaptic complex by 'simple rotation' of DNA-linked resolvase promoters. Surprisingly, DNA knots are recombined efficiently in the absence of supercoiling, whereby the rate of recombination increases with the number of irreducible DNA segment crossings, or nodes, within each substrate knot. Recombination products contain three knot nodes less than substrates, suggesting that a reduction in writhe drives the reaction. However, the proposed protomer rotation model predicts that writhe is not altered during the process of strand transfer but, instead, is reduced only when a synaptic complex disassembles after strand exchange. I present evidence that recombination of knotted and of linear substrates coincides with a disassembly of synaptic complexes. The results lead to a variant model for strand exchange on non-supercoiled substrates in which a specific disassembly of the synaptic complex, triggered by a reduction in writhe, guides the cleaved DNA into the recombinant configuration.


Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster. We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.


Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses. The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.


Two distinct subforms of the retrotransposable DRE element in NC4 strains of Dictyostelium discoideum. Approximately 2% of the Dictyostelium discoideum genome consists of multiple copies of a retrotransposable element termed DRE (Dictyostelium Repetitive Element). These elements have always been found integrated in a position and orientation-specific manner 50 +/- 4 nucleotides upstream of the coding region of tRNA genes (tDNAs). An intact DRE is 5.7 kb long. It carries an extensive coding region flanked by non-identical long terminal repeats (LTRs), composed of three distinct modules A, B and C. The left LTR proximal to the tRNA gene contains one or several A-modules followed by a single B-module (AnB). By contrast, the right LTR is composed of a B-module followed by a C-module (BC). Approximately 50% of the DRE elements in NC4 derivatives of D. discoideum are structurally different from the 5.7 kb DRE described above. They carry the following alterations: a) a 3.1 kb deletion in the coding region; b) two small deletions of 8 and 29 nucleotides in the B-module of the right LTR; c) a 72 bp deletion in the B-C junction; and d) three distinct point mutations within the A-module of the left LTR. The deletion in the open reading frame encompasses the putative coding regions for reverse transcriptase adn integrase. At least 60 copies of this smaller 2.4 kb DRE subtype are found in the genome of D. discoideum NC4 strains associated with tRNA genes. Thus, inspite of their lack in reverse transcriptase and integrase those 2.4 kb elements are presumably transposable and at least all isolated copies are found exclusively in the proximity of tRNA gene loci. The enzymes needed for their replication and transposition are likely to be provided by the intact 5.7 kb DREs.


A cyclic AMP response element is involved in retinoic acid-dependent RAR beta 2 promoter activation. Activation of the retinoic acid receptor (RAR) beta 2 promoter is known to be mediated by a RA response element located in the proximity of the TATA-box. By deletion studies in P19 embryonal carcinoma cells we have analyzed the RAR beta 2 promoter for the presence of additional regulatory elements. We found that the cyclic AMP response element-related motif, TGATGTCA at position -99 to -92, is able to enhance RA-dependent RAR beta 2 promoter activation. In addition we demonstrate that this element, designated CRE-beta 2, is functionally active as a CRE since it can bind members of the CREB/ATF transcription factor family and, moreover, mediates the stimulatory effect of cAMP on RA-dependent RAR beta 2 promoter activation in human foetal kidney 293 cells.


Morphine-induced modulation of calcitonin gene-related peptide levels. Calcitonin gene-related peptide (CGRP) is a novel calcium-modulatory product of the gene that encodes for calcitonin. Acute administration of morphine decreases levels of CGRP in rat corpus striatum. Tolerance to morphine did not alter the levels of CGRP in any brain region or in the spinal cord of the rat. CGRP did not alter the tolerance to the antinociceptive effects of morphine. Chronic naltrexone increased the levels of CGRP in the hypothalamus. Concurrent chronic administration of naltrexone plus morphine raised the levels of CGRP in the medulla, midbrain, and spinal cord. CGRP enhances naloxone-precipitated withdrawal jumping in mice. In rats, during withdrawal the levels of CGRP were tripled in the corpus striatum and significantly reduced in the hippocampus and hypothalamus. In the corpus striatum, CGRP enhances forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation when such accumulation is suppressed (as with the chronic opiate administration), but conversely depresses forskolin-stimulated cAMP accumulation under normal conditions (as with chronic vehicle administration). These data are consistent with the hypothesis that CGRP acts as a modulatory peptide in opiate-sensitive systems and tonic opioid control of CGRP levels exists in brain.


Microstructural analysis of the effects of THIP, a GABAA agonist, on voluntary ethanol intake in laboratory rats. The effects of GABAA agonist THIP on the acquisition of voluntary ethanol intake and the pattern of food and water consumption were examined through the use of a computer-controlled data acquisition system. Twenty male Long-Evans rats were randomly assigned to two groups, one of which received THIP (16 mg/kg, IP) and the other an equal volume of saline. Subjects were presented with a free choice of ethanol and water immediately following drug injections, which occurred every other day. The initial concentration of ethanol presented was 2% and was increased by increments of 2% following the second presentation of each concentration, up to a maximum concentration of 10%. Subjects treated with THIP consumed significantly greater amounts of ethanol than did saline controls. A microstructural analysis of bout patterns suggested that the increased consumption of ethanol was a function of an increase in the size, duration, and frequency of ethanol drinking bouts. Food intake was also attenuated by THIP treatment. The results indicated that the decrease in total food intake was a function of a decrease in the frequency of the food bouts. However, in contrast to that observed for ethanol intake, the size and duration of the food bouts were unchanged. The qualitatively different patterns in the microstructure of consummatory behavior for ethanol and food following THIP treatment would suggest that differential mechanisms may mediate the food and ethanol effects observed in the present study. In addition, the differential effects of THIP on ethanol consumption relative to water would suggest that GABAA manipulations may play a role in influencing the acquisition of voluntary ethanol drinking.


Maternal marijuana smoking alters respiratory timing in the fetal lamb. The effect of single and repeated maternal marijuana smoke exposure on fetal breathing movements (FBMs) was investigated in 13 fetal lambs in the third trimester. These animals were surgically instrumented for long-term intrauterine recording of diaphragmatic electromyogram (EMG). Maternal inhalation of marijuana smoke [1.84% tetrahydrocannabinol (THC)] increased FBMs and resulted in a more continuous and regular breathing pattern. There was a significant increase in the number of breaths/h (p < 0.01) and the incidence of FBMs (p < 0.001) in the second hour. Breathing activity returned to presmoke level by the third hour. Duration of the longest breathing epoch was significantly increased from 16.8 +/- 3.3 min to 31.9 +/- 5.2 min (p < 0.005). Instantaneous breathing rate was much more stable in the second hour after marijuana exposure (p < 0.01). Inhalation of placebo smoke did not result in any significant change in either overall breathing activity or continuity and stability of the breathing rate. The effects of marijuana smoke on fetal breathing were not observed after repeated smoke exposure. These results suggest that tolerance develops rapidly to the respiratory stimulating effect of marijuana smoke in the fetus.


Molecular modelling studies on the digitalis binding site of the Na+/K(+)-ATPase. Using molecular modelling methods, several digitalis-unlike compounds such as chlormadinol acetate and cassaine, which bind to the digitalis receptor and inhibit the Na+/K(+)-ATPase were compared with cardenolides as a standard. The interaction energies of this group of compounds with various probes such as a methyl group or a NH-amid group were calculated using GRID and compared using GRAD. A pharmacophore model was derived, which describes all corresponding inotropic substrates. On this basis and including experimental knowledge on the Na+/K(+)-ATPase a receptor model was developed.


[Bacterial polysaccharides of polymyxan 88A. Basic characteristics and extent of possible uses]. A new high-viscous polysaccharide polymyxan from Bacillus polymyxa 88A is described. Polymyxan consists of an acid high-viscous polysaccharide (Mw 1-10 MD) and a neutral low-viscous polysaccharide (Mw 100-300 kD), which is a glucomannan containing equal amounts of monosaccharides and traces of uronic acids. The acid high-viscous polysaccharide consists of 36% glucose, 36% mannose, 7% galactose and 21% glucuronic acid. Data are presented on the application of polymyxan in baking industry and for preparation of drilling muds.


On the protonophoric activity of oxazolomycin. By the use of an artificial lipid membrane model we have shown that the antibiotic oxazolomycin (Streptomyces albus) is an effective protonophore at pH < 7.0 but conveys both protons and monovalent cations such as potassium at pH > 7.5 as a passive carrier. The ionophoric properties are suggested to be correlated with its antibacterial, antiviral, and cytotoxic activities.


Unique metabolites of eicosapentaenoic acid interfere with corpus luteum function in the ewe. Experiments were conducted to determine the in vivo and in vitro effects of metabolites of eicosapentaenoic acid on ovine luteal function. Injection of 750 micrograms methyl eicosapentaenoic acid (EPA) or methyl 12(R),13(S)-dihydroxyeicosapentaenoic acid (12,13-diHEPE) into the ovarian artery of ewes on day 10 of the estrous cycle caused a reduction in serum concentrations of progesterone by 48 h posttreatment compared with levels of this steroid in arachidic acid-treated controls (p < 0.005). Although mean serum concentrations of progesterone in methyl EPA-treated ewes during the remainder of the cycle did not differ from those in control ewes, levels in methyl 12,13-diHEPE-treated ewes remained significantly suppressed. Duration of the estrous cycle did not differ among treatment groups (p > 0.05), but more of the methyl 12,13-diHEPE-treated animals (3/5) had exhibited estrus within 3 days after injection than methyl EPA-treated (1/5) or control ewes (0/5). Slices of corpus luteum removed from ewes on day 10 of the estrous cycle were incubated with arachidic acid (controls), 12,13-diHEPE or docosatetraenoic acid (DTA). Regardless of fatty acid treatment, all tissues retained the ability to produce basal levels of progesterone during subsequent incubation. Luteal slices previously exposed to arachidic acid or DTA exhibited an increase in progesterone production in response to subsequent treatment with LH (p < 0.05). In contrast, luteal slices incubated with 12,13-diHEPE did not respond to LH with a significant increase in production of this steroid above that observed in controls. All tissues displayed a marked increase in progesterone synthesis upon treatment with 8-Br-cAMP relative to incubation of tissue alone (p < 0.001). Subcellular distribution of [14C]-12,13-diHEPE in luteal cells after incubation revealed that the majority of the fatty acid was associated with the plasma membrane. These data suggest that metabolites of eicosapentaenoic acid with hydroxyl groups on adjacent carbon atoms interfere with luteal function in the ewe, perhaps in part by altering luteal response to LH.


Effect of BI-L-239, A-64077 and MK-886 on leukotriene B4 synthesis by chopped guinea pig lung and on antigen-induced tracheal contraction in vitro. The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.


Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding. Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.


[Surveillance of adverse effects following immunization (AEFI)]. The meeting of Working Group on Adverse Events Following Immunization (AEFI) was organized in Paris on February 5-6-7 1992. Reporting and Surveillance of AEFI in the European countries was advised to be introduced in each country as the part of monitoring system in this matter. Channels for reporting system of AEFI should be similar to channels for reporting system of communicable diseases or to system of events following drugs. Reporting and surveillance system of AEFI in Poland as well as proposition for improving of activities in this field.


[The magnetic resonance of small hepatocarcinoma. A comparison with echography, computed tomography, digital angiography and computed tomography with lipiodol]. Diagnostic techniques as a whole and periodic ultrasonography (US) in particular frequently allows tumors < 3 cm (small hepatocellular carcinomas) to be detected in patients suffering from liver cirrhosis. Multifocal diseases are a major limitation to surgery. Recently, MR imaging has shown its capabilities in the diagnosis of small hepatocellular carcinomas. In our study the diagnostic value of MR imaging was compared with that of US, of pre- and post-contrast CT, of digital angiography and of CT after lipiodol injection (Lipiodol CT). The morphologic and signal intensity MR features of small hepatocellular carcinomas were investigated. Fifteen cirrhotic patients with 31 nodules of hepatocellular carcinoma < 3 cm were examined. All patients were studied with US, MR imaging, angiography and Lipiodol CT; 12/15 patients underwent CT. Histologic confirmation was obtained in 12 nodules (2 at surgery and 10 by means of percutaneous biopsy); in the extant 19 cases the diagnosis was made by combining US, CT, MR, angiographic and lipiodol-CT findings; in 9 tumors < 1 cm Lipiodol retention one month after angiography was considered as diagnostic. MR imaging detected 21/31 nodules (63%), US 22/31 (66.6%), CT 12/24 (50%), angiography 24/31 (74%), lipiodol CT 29/31 (92.5%). Mc Nemar test showed no difference in sensitivity between MR imaging and CT, MR and angiography, MR and US, lipiodol CT and angiography; however, the differences between the detection rates of MR imaging and Lipiodol CT and CT and lipiodol CT and US were statistically significant (p < 0.05). The difference in sensitivity between the detection rates of lipiodol CT and US was just above the threshold value which is usually considered significant (p = 0.065). One false positive was observed on US and none with MR, CT, angiography and lipiodol CT. On Se T1-weighted images 18 nodules were hyperintense, 2 isointense and 2 hypointense; on proton-density images 14 nodules were hyperintense, 7 isointense and none hypointense. On SE T2-weighted images 18 nodules were hyperintense, 3 isointense and none hypointense. A pseudocapsule was seen in 10/17 nodules (58%), especially on T1-weighted images. Accuracy and limitations of each technique and morphologic and signal intensity MR findings of small hepatocellular carcinoma are discussed. We believe that US is still the best diagnostic technique for the screening of hepatocellular carcinomas in cirrhotic livers.(ABSTRACT TRUNCATED AT 400 WORDS)


[Chemoembolization in the therapy of hepatocarcinoma. A 3-year experience]. This study was aimed at evaluating the efficacy of chemoembolization (CE) to improve survival in patients with hepatocellular carcinoma (HCC). Our results were compared with the natural history of HCC. Sixty-two consecutive patients with HCC in Okuda's stages I and II underwent CE. Forty-seven patients were treated with CE alone; 9 patients had CE prior to surgery, and 6 patients had it after surgery because of recurrent HCC. One hundred and nine CEs (mean: 1.8 CEs/patient) were performed with Lipiodol UF, epirubicin and gelatin sponge. Actuarial survival was calculated considering Okuda's stage, neoplasm size, and evidence of pseudocapsule. The mean cumulative survival of the 47 patients treated with CE alone was 13.2 months; survival (+/- SE) at 12, 24 and 36 months was 0.75 (+/- 0.07), 0.46 (+/- 0.10) and 0.28 (+/- 0.12). Survival was not affected by Okuda's stage, neoplasm size, evidence of pseudocapsule (p > 0.05). Nevertheless, the patients with early HCC had better prognosis. Eighteen patients (42.9%) died during follow-up, 12 of whom (66.7%) from hepatic failure. The mean survival of patients with recurrence of HCC after surgery was 41 months (range: 24.8-74.9 months) since initial diagnosis of HCC, and 14.8 months (range: 7.1-29.6 months) since diagnosis of recurrence. Two of these patients died from hepatic failure. All the patients who underwent also surgery after CE are still alive (mean survival: 14.7 months). Histologic findings of resected specimens revealed viable neoplastic cells in all cases. Twenty-one major complications (20.2%) occurred in 18 patients (29%); the outcome of complications was favorable in all but one patient who died from sepsis. CE is a reliable and safe treatment for unresectable HCC. Small HCCs should be preferably treated with surgery or, alternatively, with percutaneous alcohol injection.


[The proton radiotherapy of melanoma of the uvea. The technic, methodology and first clinical observations]. Since June 1991, a Cyclotron unit with a proton beam of 65 MeV has been working at the Institute Antoine Lacassagne in Nice, France. An international cooperative group has been founded to guarantee the thorough exploitation of the facility through complete cooperation. From June 17 to December 20, 1991, 47 patients with uveal melanoma were treated; 4 of them entered the study by means of the Italian team and were treated in cooperation with the Cyclotron staff. Treatment modalities and preliminary observations are described.


Comparison of doxycycline and a chemically modified tetracycline inhibition of leukocyte functions. Tetracyclines (Tc's) have the ability to inhibit neutrophil (PMN) functions which may be of value in the treatment of neutrophil-driven pathologic processes. However, long term use of Tc's frequently lead to emergence of antibiotic resistant strains of bacteria. A chemically modified analogue of Tc, 4DTc, having minimal antibiotic activity was compared to Dc, a member of the Tc family, for its ability to inhibit neutrophil functions. 4DTc was significantly less effective at inhibiting PMN superoxide synthesis, PMA-induced degranulation and PMN-mediated RBC lysis than was Dc. 4DTc and Dc were equally as effective inhibiting monocyte, but not PMN, adherence to gelatin-coated surfaces. When incubated in media containing varying 4DTc or Dc concentrations, Dc accumulated in RBC's and PMN's at levels 3 times greater than that found for similar media levels of 4DTc. The data suggest that the lesser ability of 4DTc to inhibit several PMN functions as compared to Dc may be related to its reduced intracellular accumulation. It also suggest that Tc inhibition of monocyte adherence may be more influenced by extracellular than intracellular processes.


Effect of some novel ethylenediamine and ethanolamine derivatives on carrageenan-induced inflammation. Correlation with antioxidant activity and structural characteristics. The effect of eight ethylenediamine and ethanolamine derivatives on inflammation was investigated in the carrageenan-induced rat paw edema model. The ability of these compounds to inhibit superoxide anion radical (O2-.) formation in vitro was also examined using the xanthine-xanthine oxidase system. Almost all of these substances were found to possess anti-inflammatory activity. This action can be well correlated with their reported capacity to inhibit microsomal membrane lipid peroxidation, while they demonstrated negligible effect on O2.- generation. The above actions appear to depend on some structural characteristics, particularly in the aromatic series of compounds.


Increased prostaglandin E2 and cAMP phosphodiesterase levels in Kaposi's sarcoma--a virus against host defense mechanism. In Kaposi's sarcoma tissue, prostaglandin E2 (PgE2) levels and cAMP phosphodiesterase levels were found to be higher than in surrounding normal tissue. We have shown earlier that PgE2 suppresses interferon (IFN) alpha production. High levels of cAMP phosphodiesterases result in low cAMP levels. Thus, this phenomenon may be involved in altered immunologic resistance, growth and differentiation.


Are Wistar rats not susceptible to organophosphate-induced delayed neurotoxicity? Male Wistar rats were sacrificed 12 weeks after single exposure to various organophosphate compounds. Peripheral nerves and skeletal muscles were examined light microscopically for the occurrence of a delayed polyneuropathy. Although unequivocal morphological hallmarks of OPIDN had been demonstrated in other rat strains using similar doses of TOCP or mipafox, we were unable to demonstrate any abnormality with these compounds. Normal findings were also obtained with fenthion, the delayed neuropathic potential of which is debated, and with paraoxon or parathion, which are both highly unlikely to cause OPIDN. These data indicate that the Wistar rat strain is highly likely to be resistant to OPIDN.


Therapeutic effects of oral rebamipide and in combination with cimetidine on experimental gastritis in rats. The present study evaluated the therapeutic effects of rebamipide alone and in combination with cimetidine on experimental gastritis established by the administration of 5 mM sodium taurocholate (TCA) for 6 months in rats. Morphological and biochemical analyses were performed to determine the effects of rebamipide, administered alone or in combination with cimetidine, on chronic, atrophic and erosive gastritis. Rebamipide and cimetidine were administered to rats as a dietary admixture for 4 weeks after withdrawal of TCA. Rebamipide dose-dependently reduced the total length of the erosion, normalized the mucosal thickness and increased the number of parietal and total cells, and tended to reduce interstitial infiltration of inflammatory cells and proliferation of collagenous fibers. Moreover, histochemical and biochemical studies also showed rebamipide to be effective. Rebamipide increased the PAS-positive mucus and normalized the reduced gastric mucosal SOD activity. The therapeutic effect of rebamipide on the experimental gastritis was enhanced by the combined use of cimetidine. These results suggest that rebamipide has a therapeutic effect on chronic atrophic erosive gastritis induced by TCA, and that the mechanism of the therapeutic effect is partially due to the increase in PAS-positive mucus and gastric mucosal SOD activity. Furthermore, the enhanced effect of the combination therapy of rebamipide with cimetidine was considered to be due to the actions of both cimetidine and rebamipide.


Superoxide dismutase and glutathione in the gastric mucosa of patients with chronic liver disease. The total activity of superoxide dismutase and glutathione peroxidase and the tissue content of Cu,Zn-superoxide dismutase, glutathione, and lipoperoxides in the gastric mucosa were determined in patients with chronic liver disease and in healthy controls. The mean levels of Cu,Zn-superoxide dismutase in liver cirrhosis and in hepatocellular carcinoma with cirrhosis were significantly reduced compared to controls (32.0 +/- 4.4, and 35.8 +/- 2.2, vs 44.6 +/- 2.2 ng/mg protein, p < 0.01). Mucosal levels of glutathione were significantly lower in chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma with cirrhosis than in controls (9.7 +/- 2.1, 8.9 +/- 2.3, and 11.0 +/- 3.4, vs 23.6 +/- 4.7 nmol/mg protein, p < 0.05). However, there were no significant differences between chronic liver disease and controls in the activity of gastric superoxide dismutase and glutathione peroxidase. Gastric lipoperoxide concentrations were significantly higher in chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma with cirrhosis than in controls (0.56 +/- 0.07, 0.50 +/- 0.12, 0.50 +/- 0.05 vs 0.18 +/- 0.03 nmol/mg protein, p < 0.05). These results suggest that the concentrations of gastric mucosal antioxidants were decreased in chronic liver disease, and that these changes may be responsible for the higher frequency of gastric mucosal lesions observed in patients with chronic liver disease.


Transforming growth factor-beta (TGF-b) induced phosphorylation of the myristoylated alanine rich C kinase substrate (MARCKS) protein in ovarian granulosa cells is modulated by follicle stimulating hormone (FSH). The mechanism by which TGF-b1 affects granulosa cell physiology as well as the modulation of TGF-b1 activity by FSH are not understood. We tested the hypothesis that TGF-b1 exerts its effects on granulosa cells via activation of protein kinase C (PKC). Immunoprecipitation of the MARCKS protein from 32P labeled rat granulosa cells was used to assay PKC activation. 20 minute treatment with TGF-b1 (8 ng/ml), forskolin (30 microM), and TPA (200 nM) all caused an increase in MARCKS phosphorylation as quantified by densitometric scanning. FSH did not increase MARCKS phosphorylation above control levels while exposure of cells to both FSH and TGF-b1 (10 ng/ml) decreased phosphorylation of the MARCKS protein to control levels. These data suggests that (1) TGF-b1 signal transduction in rat granulosa cells may partially involve phosphorylation of the MARCKS protein; and, (2) in granulosa cells FSH can modulate TGF-b1 induced MARCKS phosphorylation.


Antilipoxygenase activity of amyrin triterpenes. Triterpenes--alpha-amyrin acetate, beta-amyrin acetate and beta-amyrin were tested for their effects on the synthesis of 5-lipoxygenase products in human neutrophils. All the triterpenes reduced 5-HETE synthesis without effect on LTB4 synthesis. The relative effects suggest that 5-HETE inhibition can explain the antiarthritic activity possessed by these compounds.


Sex difference in hepatic microsomal aldehyde oxygenase activity in different strains of mice. Hepatic microsomal oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) and 9-anthraldehyde (9-AA) to the corresponding carboxylic acids was investigated using six strains of male and female mice (ddN, ddY, C57BL, DBA, C3H and ICR). No significant sex difference was observed in the activity toward 11-oxo-delta 8-THC except for ICR, whereas the activity toward 9-AA was significantly higher in female than in male of ddN, C57BL, DBA and C3H mice. The present study suggests that female specific form(s) of cytochrome P450 may be responsible at least in part for the microsomal oxidation of 9-AA, but not that of 11-oxo- delta 8-THC.


Sleep architecture and sleep apnea in patients with Cushing's disease. Patients with Cushing's syndrome (CS) frequently have sleep complaints. We evaluated sleep polysomnographically in 22 patients, including 17 with pituitary-ACTH-dependent Cushing's disease (CD) and five with CS from an adrenal tumor. Data were compared to healthy controls of comparable age. Seven patients (32%) demonstrated at least mild sleep apnea (> or = 9.4 events/hour), and four of 22 (18%) had > or = 17.5 events/hour. The apneic CD and CS patients had a trend for a greater complaint of excessive daytime sleepiness. Both apneic and nonapneic groups had considerable snoring and obesity. The electroencephalographic (EEG) sleep of nonapneic patients was compared to that of normal subjects. Nonapneic CD patients differed strikingly from healthy volunteers in sleep continuity and architecture, demonstrating lighter, fragmented sleep. Rapid eye movement (REM) sleep in CD patients bore many similarities to the sleep of patients with major depression, with REM latency being significantly shortened and REM density significantly increased. Continued examination of EEG sleep in CD patients may shed light on similarities in pathophysiology between CD and major depression, disorders which are characterized by both a dysfunction of the hypothalamic-pituitary-adrenal axis and alterations in mood.


Effects of short-term treatment with an H+,K(+)-ATPase inhibitor (B8301-078) on enterochromaffin-like (ECL) cells in rat fundic mucosa. In the present study, female Sprague-Dawley rats were treated orally over 2, 14, and 29 days with 50 mg/kg/day of the H+, K(+)-ATPase inhibitor B8301-078, a substituted benzimidazole. The endocrine cells in the fundic mucosa were quantified by light microscopy with the aid of Grimelius silver staining and examined by electron microscopy. After 2 days of treatment, the number of argyrophilic Grimelius-positive cells (GPC) was clearly reduced compared to the controls, whereas after 14 and 29 days, hyperplasia was observed. Electron microscopy showed that there was only an apparent decrease in GPC density after 2 treatments. This reduction was due to the massive degranulation of the enterochromaffin-like (ECL) cells and the resultant decrease in stainability with silver. After 14 and 29 days the granular depletion was less pronounced. The cells were, therefore, detectable again by light microscopy.


Increase in plasma thrombomodulin and decrease in plasma von Willebrand factor after regular radiotherapy of patients with cancer. In this study, we investigated plasma levels of thrombomodulin (TM) and von Willebrand factor (vWF) in 51 patients suffering from cancer or tumor undergoing 60 cobalt radiotherapy. Plasma TM and vWF antigen were measured by immunoradiometric assay and ELISA, respectively. During radiotherapy, an increase in plasma TM in patients was observed, which was radiation-dose dependent and there was a positive correlation between plasma TM level and radiation doses. However, the level of plasma vWF in the patients was decreased during radiotherapy and there was an inverse correlation between the amount of plasma vWF and radiation doses. Our data indicate that plasma TM is an useful molecular marker for early detection of radiation injury to endothelial cells in patients undergoing radiotherapy.


Study on neutralization of low molecular weight heparin (LHG) by protamine sulfate and its neutralization characteristics. The neutralizing effects of protamine sulfate (PS) on anticoagulant activities of low molecular weight heparin (LHG) and conventional sodium heparin (Heparin) were investigated. The in vitro anti-factor Xa and APTT-prolonging activities of Heparin were almost completely neutralized by PS, whereas the activities of LHG remained partially intact in the presence of PS. Crossed immunoelectrophoresis of antithrombin III (AT III) and affinity chromatography of LHG- and Heparin-cellulose showed that AT III was substantially less dissociated from its binding to LHG than to Heparin in the presence of PS. As in vitro, the in vivo anticoagulant activities of Heparin administered i.v. to rabbits were almost completely neutralized by PS, while the anti-factor Xa and APTT-prolonging activities of LHG remained partially intact in the presence of PS. The thrombin time-prolonging activity of LHG, however, was completely inhibited by PS. Since the bleeding effect of Heparin or LHG is considered mainly due to its anti-thrombin activity, PS may be used as an agent to neutralize LHG, as in the case of Heparin, when bleeding happens to occur during LHG treatment.


Effect of cimetidine on the density of adrenergic alpha-1 and alpha-2 receptors in the rat whole prostate. In order to study the antiprostatic effect of cimetidine, we measured adrenergic alpha-1 and alpha-2 receptors of the whole prostate of cimetidine-administered rats using radioligand binding techniques. Saturation experiments using 3H-prazosin and 3H-yohimbine revealed that there were significant amounts of alpha-1 and alpha-2 receptors in the rat whole prostate. There were more alpha-1 receptors than alpha-2 receptors in both the control and treated groups. In the treated group, the density of alpha-1 receptors showed little change, while that of alpha-2 receptors showed a significant decrease in comparison to the control group without changes in KD values. These data suggest that cimetidine affects the prostatic alpha receptors and consequently decreases the density of adrenergic alpha-2 receptors in the prostate.


The quantitative difference in adrenergic alpha-1, alpha-2 and beta receptors between the prostatic and epididymal ends of dog vas deferens. In order to study the regional differences in the distribution of adrenergic receptors of the vas deferens, we measured the amounts of adrenergic alpha-1, alpha-2 and beta receptors in prostatic, intermediate, and epididymal portions of the dog vas deferens using radioligand binding techniques. Saturation experiments with 3H-prazosin, 3H-yohimbine, and 3H-dihydroxy-alprenolol demonstrated that there were significant amounts of adrenergic alpha-1, alpha-2, and beta receptors, respectively, in these three portions. alpha-1 receptors existed most densely in the whole vas deferens. The prostatic portion was found to have the largest amounts of alpha-1 and alpha-2 receptors among three portions studied. beta-Receptors were distributed most densely in the intermediate portion. alpha-1, alpha-2, and beta adrenergic receptors show different distribution patterns throughout the whole length of vas deferens.


The effect of tributyltin chloride on vascular responses to atrial natriuretic peptide. The effects of tributylin chloride (TBT) on vascular smooth muscle responses to norepinephrine, nitroprusside (SNP) and atrial natriuretic peptide (ANP) were studied in isolated aortic rings of rats. TBT did not interfere with norepinephrine-induced contraction or SNP-induced vasorelaxation. However, TBT produced a dose-dependent inhibition of ANP-induced vasorelaxation. Inhibition was not observed with inorganic tin chloride, SnCl2. The inhibition of vasorelaxation was accompanied by a parallel inhibition of ANP-induced cGMP generation. SNP-induced generation of cGMP was not affected by TBT. TBT did not interfere with binding of ANP to its receptor in bovine adrenal glands suggesting that the effects of TBT were mediated by direct interaction with membrane-bound guanylate cyclase.


The in vivo effects of cyanide and its antidotes on rat brain cytochrome oxidase activity. The in vivo effects of sodium cyanide and its antidotes, sodium nitrite, sodium thiosulfate and 4-dimethylaminophenol (DMAP), as well as the alpha-adrenergic blocking agent phentolamine, on rat brain cytochrome oxidase were studied. The course of inhibition was time-dependent and a peak of 40% was attained between 15 and 20 min after the s.c. injection of 1.3 LD50 (12 mg/kg) of cyanide. Pronounced dose-dependence was observed in the inhibition of the enzyme, at this relatively low, but lethal dose. Further observation was impossible because of rapidly lethal effects of cyanide. In animals artificially ventilated with room air, observation was possible up to 60 min. However, maximum inhibition was also 40%. When antidotes were applied 30 min after 20 mg/kg of cyanide, marked reactivation of cytochrome oxidase activity was observed with all antidotes (particularly with thiosulfate) except for phentolamine which had no effect. Prevention of methemoglobin forming with toluidine blue did not affect the reactivating ability of nitrite or DMAP, thus suggesting more complex protective mechanisms then simple methemoglobin formation. The high efficacy of thiosulfate may be attributed to its rhodanese catalyzed, direct binding to free blood cyanide, leading thus to its dissociation from cytochrome oxidase. The theory that cytochrome oxidase inhibition is a basic mechanism of cyanide toxicity could not be disproved.


Mobilization of lead in mice by administration of monoalkyl esters of meso-2,3-dimercaptosuccinic acid. The following six monoalkyl esters of meso-2,3-dimercaptosuccinic acid (DMSA) were synthesized and evaluated for relative activities in mobilizing lead from kidneys and brains of lead-bearing mice: n-propyl (Mn-PDMS), i-propyl (Mi-PDMS), n-butyl (Mn-BDMS), i-butyl (Mi-BDMS), n-amyl (Mn-ADMS) and i-amyl meso-2,3-dimercaptosuccinate (Mi-ADMS). DMSA was used as a positive control. When each was administered intraperitoneally (i.p.) as a single dose of 2.0 mmol/kg, DMSA lowered the kidney lead concentration 52%, while the monoesters effected reductions of 54-75%. Mn-ADMS was toxic at this dose. DMSA lowered the brain lead level 20% when given as a single dose, while the monoesters conferred reductions of 64-87%. When given as 5 daily i.p. injections at 0.5 mmol/kg, DMSA reduced the kidney lead concentration 45%, while the monoesters caused reductions of 56-73%. DMSA lowered the brain lead concentration 35% on the 5-day treatment regimen, while the monoesters evoked reductions of 59-75%. Mi-ADMS was equally effective when given orally or i.p. The i.p. LD50 value of this analog in mice is 3.0 mmol/kg, a value which lies between the reported LD50 doses of DMSA (16.0 mmol/kg) and dimercaprol (1.1 mmol/kg). It is suggested that the ability of these monoesters to cross cell membranes may account for their superiority to DMSA in mobilizing brain lead in this animal model.


Why is the genome variable? Insights from Drosophila. The analysis of variation in DNA restriction maps and DNA sequence in natural populations of Drosophila melanogaster and related species has revealed a remarkable richness of diversity. This review describes some of the results of population genetic studies of this variation that are beginning to reveal how interactions between natural selection, genetic drift, mutation rate, recombination rate and population size have contributed to the observed patterns.


[The use of DNA hybridization in situ for identifying chromosomal rearrangements in the karyotyping of cell lines]. A complex karyological analysis of three human cell lines (PLC-PRF-5, MT-4 and U937) was carried out that involved traditional methods of G-, C-banding and silver staining, in addition to the in situ hybridization technique using 4 alpha-satellite DNA probes: DNA specific for centromeric regions of chromosome 11, 6, 13, and 21, and 14 and 22. The application of this additional method allowed to identify, prove or detalize the structure of 13 markers in PLC-PRF-5 cells, 1 marker in MT-4 cells, and 3 markers in U937 cells. The results show that the in situ hybridization method would be successful in cell line karyotyping for a more objective identification of some markers difficult for analysis.


Regional difference in functional roles of cAMP and cGMP in lower urinary tract smooth muscle contractility. The spontaneous contractile force of muscle strips isolated from rabbit urinary bladder dome, base and urethra was dose-dependently inhibited by isoproterenol, an adenylate cyclase activator through beta-adrenoceptors and also by sodium nitroprusside, a guanylate cyclase activator. The relaxation response by isoproterenol was biggest in urinary bladder dome. Percent relaxation to 10(-4) M isoproterenol was 73.6% in bladder dome, 56.1% in bladder base, and 44.1% in urethra. The relaxation response by nitroprusside was biggest in urethra. Percent relaxation to 10(-4) M sodium nitroprusside was 34.8% in bladder dome, 51.2% in bladder base, and 63.2% in urethra. Cyclic adenosine monophosphate (cAMP) accumulation by isoproterenol was greatest in dome. cAMP levels increased by 150% in bladder dome, by 74% in bladder base and by 80% in urethra after 1 min over basal levels to become stable for 5 min. Cyclic guanosine monophosphate (cGMP) accumulation by sodium nitroprusside was greatest in urethra. cGMP levels increased by 445% in urethra after 1 min over basal levels and by 320% in dome, by 380% in base and by 1,100% in urethra after 5 min over basal levels. Dibutyryl cAMP relaxed the dome, base and urethra. 8-bromo cGMP also relaxed them. These results suggest that the role of cGMP is mainly related to urethral relaxation, whereas the role of cAMP is mainly related to urinary bladder relaxation.


Adrenergic and cholinergic muscarinic receptors in the prostate of young and old rabbits. We measured the autonomic receptors in prostate homogenates of young (6 months) and old (4.5-5 years) rabbits in order to study the effect of age on the prostatic autonomic receptors using radioligand binding techniques. Saturation studies with 3H-prazosine, 3H-yohimbine, 3H-dihydroalprenolol and 3H-quinuclidinyl benzilate demonstrated a significant amount of alpha 1-, alpha 2-, beta-adrenergic and muscarinic cholinergic receptors in prostatic tissues of both young and old rabbits. There were more alpha 1- and alpha 2-receptors in the prostate of old rabbits, although these differences were not statistically significant. There was no difference in the amount of beta-adrenergic and muscarinic receptors in the prostates of young and old rabbits. But there was a difference in the distribution of these autonomic receptors in the prostates of young and old rabbits. These results suggest that aging may affect the distribution of autonomic receptors, but not significantly the amount of autonomic receptors in the rabbit prostate.


Involvement of the inferior caval vein in adrenal metastasis. A patient was referred to our hospital for resection of a large renal cell carcinoma with invasion of the inferior caval vein, diagnosed as such with CT, angiography and cavography. The history mentioned partial resection of the left lung for lung carcinoma 16 months before. At operation the tumor could not be removed, the patient died because of postoperative pulmonary complications. Autopsy and histopathological examination revealed a large metastatic tumor of the previous lung carcinoma in the left adrenal gland. The clinical implications and some diagnostic methods are discussed.


Computer analysis of the amino acid sequences in gp41 of apathogenic African green monkey (AGM) virus, less pathogenic HIV-2 and highly pathogenic SIV and HIV-1 lentiviruses. The bestfit computer program was used to compare the amino acid sequence of the gp160 envelope glycoprotein of an apathogenic AGM and the pathogenic SIVAGM monkey lentiviruses. It was found that the gp120 envelope glycoproteins of these viruses resembled each other in their functional domains. However, an insert of 40 amino acids was found in the gp41 envelope glycoproteins of the pathogenic SIVAGM virus in the amino acid sequence between the membrane anchoring sequence and the carboxyterminus. The insert introduced a new "RRIR" proteolytic cleavage signal into gp41. Comparing HIV-1 gp41 to that of the pathogenic SIVAGM virus revealed that the HIV-1 sequence contains an "RR" sequence that also serves as a signal for proteolytic cleavage. Comparing HIV-2 gp41 to the apathogenic and pathogenic simian immunodeficiency viruses revealed that HIV-2 gp41 lacks the above proteolytic cleavage signal. It is hypothesized that the pathogenic human and simian immunodeficiency lentiviruses can be proteolytically cleaved at the carboxyterminus of gp41, releasing two peptides: a) an "immunodeficiency" 58 amino acid peptide and b) an IL-2-like peptide. The apathogenic AGM virus and the less pathogenic HIV-2 lack one proteolytic cleavage signal in the gp41 amino acid sequence and therefore can release only the IL-2-like peptide but not the "immunodeficiency" peptide. If indeed the pathogenic SIVAGM and HIV-1 do release an "immunodeficiency" peptide, then such a peptide can be regarded as a toxin. Immunization of healthy individuals or HIV-1 patients against the toxic effect of the viral gp41 toxic peptide might prevent damage to the immune system when the virus reactivation leads to ARC and AIDS in infected individuals. Synthetic peptides modeled according to the immunodeficiency peptide (the toxin) can be used to produce anti-toxin antibodies in healthy HIV-1 infected individuals. Such anti-toxin antibodies can be used for passive immunization of AIDS patients or for active immunization of HIV-1 positive individuals prior to ARC or AIDS.


Nucleotide sequence of the genome segment encoding nonstructural protein NS1 of bluetongue virus serotype 20 from Australia. The nucleotide sequence of the genome segment (S6) encoding the nonstructural protein NS1 of an Australian isolate of bluetongue virus serotype 20 (BTV 20) has been determined from a series of overlapping cDNA clones synthesized using two terminal 15-mer oligonucleotides as primers. The gene consists of 1769 nucleotides with an open reading frame between nucleotides 35 and 1690 encoding a protein of 552 amino acids (molecular weight 64,506 Da; net charge -2 at pH 7). Comparison of the nucleotide and deduced amino acid sequence of this genome segment with cognate segments of isolates of BTV 1 from Australia and South Africa, and BTV 10 and BTV 17 from the United States, revealed homologies of 98%, 80%, 79%, and 79%, respectively, at the nucleotide level and 98%, 90%, 89%, and 90% identity, respectively, at the amino acid level. The data indicate that the evolutionary divergence between NS1 genes of two different Australian BTV serotypes (BTV 20 and BTV 1) is less than that between isolates of the same (BTV 1) or different serotypes from different geographical locations.


Nucleotide sequence of the cDNA for porcine rotavirus VP7 gene (strain K). The nucleotide sequence of the cDNA encoding one of the neutralizing proteins VP7 of the new porcine strain K is determined. The deduced VP7 amino acid sequence of the K strain showed a high homology (93%) and a lower homology (75%) to those of the Gottfried and OSU strains, respectively. This finding suggests that strain K is more closely related to the Gottfried strain serotype 4.


[Minerals of biological origin: their structural formation and properties]. A study is presented of human tooth enamel and its changes during heating. The measurements were done by methods of electron paramagnetic resonance, roentgen structural analysis and gaseous chromatography. A new effect has been found related to changes of enamel structure during heating. It was established that at T 170 degrees C there occurs reorientation of the crystallites of hydrohylapatite that form the structure of tooth enamel as well as changes of the properties of organic layers between these crystallites. Discussed are the general principles determining the structure and properties of minerals of biological origin. It is shown that the organic structure of the biomineral effects the properties of inorganic crystallites by means of the spatial geometric factor. This factor is finally related to the fact, that superficial energy of the crystallite contributes essentially in its general energy. The authors discuss the obtained results from the viewpoint of diagnosis and new treatment methods of diseases related to the biomineral of the human body.


[Breast cancer in the catchment area of the Graz Institute of Pathology. Evaluation of morphologic parameters based on 1,510 cases]. The patient's age, tumour size, histological type and degree of differentiation as well as involvement of axillary lymph nodes are decisive for prognosis and therapy of breast cancer. Moreover these parameters reflect the achievement of early diagnosis and the surgical standard of treatment of breast carcinomas. Therefore we retrospectively reviewed 1510 cases diagnosed from 1984-1987. Non-invasive carcinomas were diagnosed in 4%. 75% of them were classified as intraductal carcinoma and 25% as lobular carcinoma in situ. 96% of the tumours were invasive at time of diagnosis. Invasive ductal carcinoma (NOS-type) was found in 70.2%, invasive lobular carcinoma in 12.3%. 3.2% of the tumours showed both ductal and lobular differentiation and 2.3% corresponded to invasive ductal carcinoma with a predominantly intraductal component. Medullary and mucinous carcinomas were detected in 2.1% and 2% of cases, respectively. Papillary carcinomas were observed in 0.9%, the frequency of other histological types was less than 1%. 44% of the tumours corresponded to UICC-category pT1, 38% to pT2, 6% to pT3 and 8% to pT4. A meaningful correlation of tumour size and axillary lymph node involvement was possible in only 906 cases, in which 10 or more lymph nodes were verified histologically. Lymph node metastases were detected in 23% of tumour category pT1 and in 47% of category pT2. PT3- and pT4-tumours metastasized to axillary lymph nodes in 77 and 86% of cases, respectively.


Prevention of viral hepatitis A: past, present and future. Before hepatitis A virus (HAV) was identified, spread of hepatitis A was prevented by public health measures. The first specific, preventive measure for hepatitis A was passive protection with standard, pooled human immune globulins. Human immune globulin contained sufficient HAV neutralizing antibodies for short-term, prophylactic passive protection and for control of the spread of local outbreaks. After many unsuccessful attempts, HAV was propagated in cell cultures and the development of vaccines for active immunization began. Formalin-inactivated, whole HAV induced protective immunity, and such formalin-inactivated hepatitis A vaccines are now being evaluated in large-scale clinical trials. HAV attenuated by serial propagation in cell culture has been used for several, live, attenuated hepatitis A vaccines and results of clinical trials are reassuring. Future approaches to protection against hepatitis A are likely to include vaccination with: hybrid viruses; hepatitis A antigen-expressing, genetically-engineered bacteria; purified hepatitis A antigens produced by molecular biological techniques and incorporated into slow or pulse-releasing systems; synthetic peptides or idiotypes.


Laboratory tests and reference reagents employed in studies of inactivated hepatitis A vaccine. Procedures to evaluate inactivated hepatitis A vaccines in volunteers have been examined. Solid-phase immunoassays were standardized with reference preparations and have been tested to measure antibody response to immunization and antigen content of vaccines. Following immunization, there was a good correlation between antibody response, determined with commercial immunoassays, and neutralization titres, as measured by the radioimmunofocus inhibition test. However, at lower titres of neutralizing antibody, the commercial immunoassay often yielded negative results. To improve the sensitivity of the immunoassay, the serum volume was increased. A fourfold increase of test serum resulted in greater sensitivity, increasing from 54 to 94%, while retaining 100% specificity. Further increases in the volume of test serum resulted in a loss of specificity. In a comparison of neutralization tests, similar titres of postvaccination sera were obtained by using the HM175/18f cytopathic strain of hepatitis A virus in a plaque reduction assay or the HM175 parental virus in the radioimmunofocus inhibition test. Use of the cytopathic virus obviates the need for radioactively labelled serum and reduces the time taken to conduct neutralization tests. The current laboratory procedures can meet the needs of large field trials of inactivated hepatitis A vaccines.


Serological approaches to distinguish immune response to hepatitis A vaccine and natural infection. Currently, the immune status of an individual exposed to hepatitis A virus (HAV) is determined by assays which measure antibodies against the capsid proteins. These assays indicate exposure to the viral capsid that could result from either infection or from vaccination. Recent data indicate that proteins from the non-structural genome region of the virus (P2 or P3), which are only produced during active virus replication, generate antibodies after clinical disease. A sub-genomic cDNA segment of HAV corresponding to the P2 region was used for in vitro transcription-translation followed by immune precipitation of the translated products under non-denaturing conditions. Serial serum specimens from experimentally infected chimpanzees and humans naturally infected with hepatitis A verified the development of antibodies to P2 proteins following infection. A serosurvey of individuals positive for antibodies to the HAV capsid (HAV AB assay, Abbott Laboratories) revealed that 50-60% of children and 16-32% of adults had no detectable antibodies to the P2 antigen by immune precipitation. These results may reflect subclinical infections resulting in a lower level of antibodies against the non-structural antigens or may represent a greater sensitivity of the competitive assay (HAV AB) used to detect capsid antibodies compared to the immunoprecipitation assay used to detect non-structural antigens.


Effect of virus strain and antigen dose on immunogenicity and reactogenicity of an inactivated hepatitis A vaccine. A randomized double-blind comparison of five killed hepatitis A vaccine preparations was carried out with eligible medical student and staff volunteers. Vaccines were prepared in M RC-5 cells and formalin-inactivated. Three monthly injections of 1 ml in the deltoid muscle were given. Group A received the CLF strain at a dose of 360 ELISA units (El.U) in 0.5 mg aluminium hydroxide (n = 35). The other groups received the HM175 strain as follows: 180 El.U in 1 mg aluminium hydroxide (to group B, n = 42), 360 El.U in 0.5 mg aluminium hydroxide (to group C, n = 40), 360 El.U in 1 mg aluminium hydroxide (to group D, n = 39) and 720 El.U in 1 mg aluminium hydroxide (to group E, n = 43). The geometric mean anti-HAV concentration (GMC) measured in mIV/ml by an ELISA method one month after each injection were: group A, 223, 480, 1635; group B, 123, 221, 649; group C, 185, 365, 1085; group D, 144, 323, 1076; group E, 229, 646, 2521. At month 6, the GMC had fallen by approximately 20%. Seroconversion as measured by ELISA was 100% in groups A and E after one injection, and 100% in all groups after three injections; after two injections, only one subject in group C was still negative. The dose effect with HM175 vaccine was significant. There was a good correlation between ELISA and neutralization (radioimmunofocus inhibition test) titres. One month after the second dose, all subjects in groups A and E had both hepatitis A virus immunoglobulin M (HAV IgM) geometric mean titre, (GMT > 5000) and IgG (GMT > 25,000) as measured by a sensitive terminal dilution ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)


Inactivated hepatitis A vaccine: a safety and immunogenicity study in health professionals. The safety and immunogenicity of an inactivated hepatitis A vaccine (HM175 strain) were evaluated in 150 seronegative health professionals. The age range was 21-65 years and the mean age was 30 years. The vaccine was administered at a dose of 720 ELISA units (EU) to 73 vaccinees at 0, 1 and 6 months, and to 77 vaccinees at 0, 1 and 12 months. The seroconversion rates were 88 and 90% in the two groups, respectively, one month after the first inoculation and 99 and 100% one month after the second inoculation. The geometric mean antibody titres were similar in both groups, exceeding 3000 mIU/ml one month after the third inoculation. The vaccine was well tolerated. The most frequent side effect was transient soreness at the site of the inoculation. No serious adverse reactions were observed. The study demonstrated that the HM175 inactivated hepatitis A vaccine was safe and highly immunogenic.


Effect of hepatitis A vaccination schedules on immune response. An inactivated hepatitis A vaccine was given to 104 seronegative volunteers aged between 19 and 60 years according to two schedules: 0, 1 and 2 months or 0, 1 and 6 months. The vaccine was well tolerated and 97 and 100% of vaccinees developed a serum antibody response following a single and two doses of vaccine respectively. Geometric mean titres increased progressively after each dose; responses following the 0, 1, 6 month schedule were significantly higher at one year but, among those tested at two years, these differences were less marked. Vaccinees, when compared with naturally infected persons, developed poor or undetectable hepatitis-A-virus-specific immunoglobulin G and A antibody responses in saliva and parotid fluid. Such differences are, however, unlikely to affect the protective efficacy of the vaccine.


Hepatitis A vaccination: schedule for accelerated immunization. Hepatitis A vaccine, strain HM175, was investigated for immunogenicity and tolerability in a prospective multicentre trial. The following vaccination schedules and antigen contents were evaluated: days 0 and 14 with 720 ELISA units (El.U) of antigen, days 0 and 28 with 720 El.U and days 0 and 28 with 360 El.U. In all study groups, the seroconversion rates following two vaccinations were between 95 and 100%. Higher geometric mean concentrations of antibody to hepatitis A virus (anti-HAV) were reached by the vaccine containing 720 El.U of HAV antigen. The vaccine was equally well tolerated in all groups. In addition, an abbreviated schedule, in which 720 El.U of HAV antigen was given on days 0 and 14, resulted in 100% seroconversion by day 28 and a level of anti-HAV that was substantially higher than that observed after passive immunization. This implies that such a vaccine could replace immune globulin administration if time permits.


Vaccination against hepatitis A: comparison of different short-term immunization schedules. A total of 114 healthy young adults were immunized with hepatitis A vaccine using different vaccination schedules. Individuals received either a single dose (group 1), two doses given simultaneously (group 2), two doses at days 0 and 14 (group 3) or at days 0 and 28 (group 4), or three doses at days 0, 7 and 21 (group 5). Two weeks after a single dose, seroconversion rates between 77 and 85% were achieved (groups 1, 3, 4). All individuals immunized with two doses within two weeks (groups 2, 3, 5) had antibodies to hepatitis A vaccine (anti-HAV positive) by week 3; these participants also showed clearly higher mean anti-HAV values (geometric mean titres, GMTs) at this time than those individuals vaccinated only once. GMTs at week 8 were 560 IU/l in group 5, 236, 339 and 428 IU/l in groups 2-4 and 102 IU/l in group 1. Of participants with anti-HAV at week 8, 82 were again tested 4 months later; all were still seropositive. Ten individuals were tested during the first three weeks at 3-4 day intervals for anti-HAV immunoglobulin M (IgM); specific IgM responses were not detectable before day 10 but were present in eight of 10 vaccinees by day 14.


Persistence of vaccine-induced antibody to hepatitis A virus. A level of 10 mIU hepatitis A antibodies/ml as measured by ELISA is believed to be the minimal protective concentration. If this level is considered, the mean persistence of vaccine induced antibodies is approximately 10-11 years after booster dose, 6-7 years if only the primary doses are given and 5-6 years if the minimal individual titre is taken into account.


Reactogenicity and immunogenicity of three different lots of a hepatitis A vaccine. To study the immunogenicity and reactogenicity of an inactivated hepatitis A vaccine, 204 healthy individuals were randomized into three equal groups, each to receive a different vaccine lot. Each subject received a total of three doses, each of 720 ELISA units of hepatitis A vaccine HM175, according to a 0 and 1 month primary vaccination schedule, with a booster dose given at month 6. Side effects were low and were < 30% after the second injection. All subjects but one had antibodies to hepatitis A virus two months after the first dose of vaccine. At month 6 all vaccinees had seroconverted. There were no differences between the three vaccine lots with respect to side effects, seroconversion rates and geometric mean titre of antibodies. We conclude that the three lots of inactivated hepatitis A vaccine are safe, well tolerated and equally immunogenic.


Clinical and laboratory observations following oral or intramuscular administration of a live attenuated hepatitis A vaccine candidate. Clinical observations made after immunising volunteers with a live attenuated hepatitis A vaccine are described. The candidate vaccine was prepared with the HM175 strain of hepatitis A virus and shown to be safe, immunogenic and efficacious in experimental animals. When the candidate vaccine was tested by oral administration in humans at increasing doses--10(4), 10(5), 10(6) and 10(7) median tissue culture infective doses (TCID50)--an antibody response was not observed at any dose. Volunteers who received similar doses by the intramuscular route developed antibody to hepatitis A three weeks after immunization with 10(6) or 10(7) TCID50. The antibody response was sustained for the 12 weeks of the observation period. All volunteers remained healthy with normal results from liver tests throughout the monitoring period. Further clinical observations of this product are in progress.


Simultaneous passive and active immunization against hepatitis A. The serum antibody response to simultaneous administration of immune globulin (Ig) and an inactivated hepatitis A vaccine was investigated in healthy volunteers who had been tested and found free of hepatitis A virus. One hundred and forty nine subjects were randomly allocated into three groups. Group 1 received three doses of hepatitis A vaccine at 0, 1 and 6 months, group 2 received 5 ml of Ig and group 3 received a combination of Ig and vaccine. In group 3 the seropositivity rate measured by enzyme-linked immunosorbent assay was 100% at day 5, month 1 and 2, 96% at month 6 and again 100% at month 7. In the group that received vaccine alone the seroconversion rates were 0, 96, 100, 98 and 100 respectively. The geometric mean titres in subjects who received passive/active immunization were about twofold lower than in subjects who received vaccine alone, indicating interference of Ig with the immune response. Despite this, the data show that simultaneous administration of hepatitis A vaccine and Ig confers both immediate protection via Ig administration and long-term vaccine-induced protection. As the antibody levels reached are about twofold lower compared to that after administration of hepatitis A vaccine alone, a booster dose may be required sooner, than if the vaccine were administered alone.


Simultaneous vaccination against hepatitis A and B: results of a controlled study. Hepatitis A and hepatitis B are endemic in many countries and must be considered as serious health risks for large parts of the world population. Simultaneous or combined vaccination against these two diseases would therefore be most advantageous. In order to investigate possible interactions between these vaccines with respect to their tolerability and immunogenicity, we conducted a randomized prospective study comparing single and simultaneous administration of the two vaccines. Three groups of healthy volunteers, each with 55 persons, were included in the study. All were negative for hepatitis A and hepatitis B markers and had normal serum liver enzyme values. Group I received hepatitis A vaccine (720 ELISA units) into the left deltoid muscle, group II received hepatitis B vaccine (20 micrograms) into the right deltoid muscle and group III received hepatitis A vaccine into the left, and hepatitis B vaccine into the right deltoid muscle. Three doses of the vaccines were administered at 0, 1 and 6 months. Local and systemic reactions were monitored by means of questionnaires. Blood samples for determination of antibody to hepatitis A virus (anti-HAV) and antibody to hepatitis B surface antigen (anti-HBs) and of serum SGOT and SGPT levels were drawn at months 0, 1, 2, 6 and 7. There were no serious general and only mild local reactions. The mean serum SGOT and SGPT values remained in the normal range in all groups. The seroconversion rates and mean geometric titres of the anti-HAV and anti-HBs antibodies were similar when the vaccines were administered separately or simultaneously. There were no significant differences between the compared groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Inactivated hepatitis A vaccine: active and passive immunoprophylaxis in chimpanzees. Studies of active and passive immunoprophylaxis were carried out in chimpanzees to determine whether a candidate hepatitis A virus (HAV) vaccine could stimulate antibody to HAV (anti-HAV) that was qualitatively similar to anti-HAV stimulated by natural infection. Normal immune globulin (Ig) was prepared from plasma obtained from human volunteers before and after vaccination with the HAV vaccine, and these preparations or commercially prepared Ig were administered to chimpanzees. Protective efficacy was compared to that obtained after vaccination of chimpanzees. As expected, pre-vaccination Ig did not protect chimpanzees against challenge with virulent hepatitis A. In contrast, chimpanzees were protected against hepatitis A by Ig prepared from volunteers who had received hepatitis A vaccine. The protection was qualitatively similar to that afforded by commercial normal Ig containing convalescent anti-HAV. The minimum protective dose of passively acquired anti-HAV was approximately the minimum dose detectable by serological means. This information will be useful in calculating minimum acceptable titres of anti-HAV in normal Ig. Whereas administration of Ig protected chimpanzees against hepatitis A pathology, it did not protect them from infection with HAV. Thus, these chimpanzees were protected by classical passive-active immunoprophylaxis. In contrast, chimpanzees actively immunized with HAV vaccine were apparently protected against both hepatitis A pathology and HAV infection. The mechanism of this complete protection is unknown but may simply represent the higher titre of anti-HAV in the vaccinated chimpanzees, compared to the passively protected animals.


Clinical manifestations and diagnosis of hepatitis A virus infection. Hepatitis A is an acute, necroinflammatory disease of the liver which results from infection by the hepatitis A virus (HAV). The mean incubation period is approximately 30 days. Although the disease is usually self-limited, the severity of illness is age-dependent. In children, hepatitis A is usually asymptomatic, while in adults, symptomatic infection is characteristic and jaundice is common. Fulminant hepatitis A is rare and is also age-dependent. The onset of hepatitis A is often abrupt and characteristic prodromal symptoms are followed, within a few days to a week, by dark urine and jaundice. Mild to moderate tenderness over an enlarged liver is usually detected. Serum alanine and aspartate aminotransferase levels usually both rise rapidly during the prodromal period, reach peak levels and then decrease by approximately 75% per week. Serum bilirubin concentrations reach peak levels later and decline less rapidly than serum aminotransferases. Nonetheless, the period of jaundice persists for < 2 weeks in approximately 85% of cases. Nearly all adult patients with clinically apparent disease experience complete clinical recovery with restoration of normal serum bilirubin and aminotransferase values by 6 months. Relapses and prolonged cholestasis are unusual manifestations of hepatitis A, and even in these circumstances, recovery is the rule and chronic hepatitis is not seen. The diagnosis of hepatitis A requires the detection of immunoglobulin M antibody to HAV in a patient who presents with, or has recently had, clinical features of hepatitis (icteric or anicteric disease) or in an individual with inapparent, asymptomatic infection in whom serum aminotransferase elevations may be detected.(ABSTRACT TRUNCATED AT 250 WORDS)


Placebo-controlled efficacy study of hepatitis A vaccine in Valdivia, Chile. A placebo-controlled, double-blind study on the efficacy of a hepatitis A vaccine (SmithKline Beecham Biologicals) was started in a region of Chile in September 1990, using hepatitis B vaccine as control. A total of 260 healthy children, 6-15 years of age, negative for antibody to hepatitis A virus (anti-HAV), antibody to HAV immunoglobulin M (IgM), hepatitis B surface antigen, and antibody to hepatitis B surface and core antigens by ELISA tests within 7 days before vaccination, were randomly assigned to two study groups: 128 children received the vaccine with a yellow label (group 1), and 132 children the vaccine with an orange label (group 2) at months 0, 1 and 6. Blood for serology and transaminase determination was drawn at months 1, 2, 6, 7 and 12. Both vaccines were tolerated equally well and no serious side effects were seen. In group 1 (presumed hepatitis A vaccine group), anti-HAV was detected (20% inhibition was used as the cut-off level) in 122 of 128 children (95.5%) tested at month 1, in 126 of 127 (99.2%) at month 2, in 126 of 127 (99.2%) at month 6 and in 126 of 126 (100%) at month 7. One anti-HAV seroconversion seen at month 1 was associated with presence of anti-HAV IgM and therefore probably represents HAV infection. Geometric mean anti-HAV concentration of the other children was 128, 342, 214 and 2301 mIU/ml at months 1, 2, 6 and 7, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Field evaluation of a hepatitis A vaccine in a Norwegian contingent to the United Nations Interim Force in Lebanon. The Norwegian population is basically seronegative regarding anti-hepatitis A antibodies. For this reason they are particularly vulnerable when exposed to hepatitis A virus when staying in highly endemic areas. Norwegian UN-personnel have so far been protected with serum immunoglobulins (Ig). Two studies are reported, one followed a vaccination schedule of 0, 1 and 2 months with a booster at month 12, while the other followed a schedule of day 0 and 14 with a booster at month 7. Both were field trials evaluating the new hepatitis A vaccine, from SmithKline Beecham Biological, in Norwegian UN soldiers serving in Lebanon. Both trials showed 100% seroconversion following the initial vaccination course, with low reactogenicity and no clinical or subclinical sign of hepatitis. The new vaccine has decisive advantages over immunoglobulins on medical, practical and economic grounds.