Amplified cellular oncogenes in neoplasms of the human central nervous system.

Significant advances have recently been made in a number of areas concerning central nervous system (CNS) neoplasia. Particularly salient are the following: (1) gene amplification is related to increasing grade of human glioma malignancy and occurs in approximately 40% of the most common and most malignant variety of glioma, glioblastoma multiforme (GBM), (2) by far the most commonly amplified gene in glioblastomas is the epidermal growth factor receptor (EGFR) gene, which is amplified in about one third of GBMs, (3) a small percentage of GBMs amplify N-myc or the novel sequence gli, (4) the EGFR gene is rearranged in at least half of gliomas in which it is amplified, and (5) EGFR gene rearrangement results in external domain deletions that yield truncated EGF receptors. Antibodies specific for the mutant EGF receptor fusion junction have been successfully produced and provide stimulating new potential avenues for tumor imaging and therapy. For pediatric CNS neoplasms, only medulloblastoma has been investigated in adequate numbers; a small percentage exhibit amplification of either the N-myc or c-myc genes.


Amplification and rearrangement of L-myc in human small-cell lung cancer.

DNA amplification of cellular proto-oncogenes is a well-established and common mechanism of oncogene activation in several types of human tumors, including the rapidly fatal small-cell lung cancer (SCLC). Approximately one fourth of primary SCLC tumors contain amplified copies of one of the three myc proto-oncogenes. Occasionally DNA amplification of the myc genes is associated with DNA rearrangements. Specifically, a novel locus named rlf is often involved in intrachromosomal L-myc rearrangements in SCLC. The structurally similar rearrangements are probably due to a highly repetitive region upstream of the L-myc gene, and result in the formation of a chimeric rlf-L-myc fusion protein. The consistent finding of the rlf-L-myc rearrangement in SCLC suggests that it may provide a selective advantage to the cells harboring it.


DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.


The mechanism of carcinogen-induced DNA amplification: in vivo and in vitro studies.

Exposure to chemical carcinogens provides a means for the enhancement of the frequency of gene amplification and for the facilitation of research into its mechanism(s). Using carcinogen-induced SV40 amplification as a model system it was shown that amplification of the viral sequences occurs via a replication-dependent mode. This process involves overactivation of the origin region and the generation of inverted repeats. Carcinogen-induced enhancement of gene amplification is triggered by cellular factors that could act in trans. An in vitro amplification system, based on extracts from carcinogen-treated cells and SV40 template sequences, was used to further characterize the amplification intermediates. The major products of overreplication in this system consist of sequences derived from the origin region. Our studies suggest that the ability to overreplicate the origin region in vitro derives from the combined action of carcinogen-induced factors that trigger overinitiation, with the inherent inability of Chinese hamster cell extracts to fully replicate large plasmid templates. The newly replicated sequences are not associated with the parental molecule and contain hairpin or stem and loop structures. Based on these findings we propose a novel replicative mechanism for DNA amplification that allows the de novo formation of hairpin structures. According to this model, an obstruction of the replication fork may cause an overturning of the DNA polymerase, followed by a template switch that leads to the use of the newly replicated strand as a template. This mode of replication results in the generation of hairpin structures which can function as precursors for the duplicated inverted repeats which are commonly observed in amplified genomes. This model is supported by our in vitro and in vivo studies. The relevance of this model for the amplification of cellular sequences is discussed.


Mutagenicity of substituted anthraquinones in the Ames/Salmonella microsome system.

Unsubstituted anthraquinone, 4 substituted anthraquinones (emodin, danthron, physcion, a new compound M-108-C) and 3 dimers (skyrin, rugulosin, rugulin) were tested using the Ames/Salmonella assay (strains TA98, TA100, TA1537 and TA102). Danthron and emodin were found to be mutagenic for TA1537 with or without metabolic activation, physcion only with metabolic activation. A significant difference was found between the mutagenic activities of emodin (16.2 His+/nmole) and danthron (6.5 His+/nmole) as well as a high specific mutagenic activity for physcion (11.6 His+/nmole). These results on structure-mutagenic activity relationships suggest that the 6-methyl group plays an important role in the mutagenic activity after metabolic activation. Furthermore, and contrary to emodin, physcion exhibited a weak mutagenic activity for TA102, probably due to the formation of a different metabolite. Such information is necessary to evaluate the potential carcinogenic hazard of these compounds.


Farm-grade chlorpyrifos (Durmet) is genotoxic in somatic and germ-line cells of Drosophila.

The mutagenic potential of Durmet, a farm-grade formulation of chlorpyrifos, was studied in the Drosophila wing mosaic and sex-linked recessive lethal tests. Larvae of the 2nd or 3rd instar carrying suitable recessive genetic markers on chromosome 3 were exposed to different concentrations of the insecticide and the frequency of induction of mutant mosaic spots on the wings was noted. The Basc technique was followed to study the induction of sex-linked recessive lethals. On the basis of the frequency of induction of mosaic wing spots and sex-linked recessive lethals, it is concluded that Durmet is genotoxic in somatic cells as well as germ cells of Drosophila.


The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster.

The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2-6 x 10(-5) per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5-25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.


Antimutagenic effect of eight natural foods on moldy foods in a high liver cancer incidence area.

Ames test procedures were used to test 8 natural food extracts for their antimutagenic activity against the mutagenic activity induced in S. typhimurium strains TA98 and TA100 by aflatoxin B1 (AFB1) or metabolic extracts from A. versicolor or A. ochraceus. The tested substances were extracted repeatedly with acetone. The revertants induced by AFB1, metabolic extracts of A. versicolor or A. ochraceus were significantly decreased when extracts of the 8 natural foods were added to the media. The results showed that these extracts had marked inhibitory effects on the mutagenic activity induced by AFB1 or metabolic extracts of the two molds and also suggested that antimutagenic substances were present in these natural foods. These experiments provide a scientific basis for the study of food substances for the prevention of carcinogenesis. It is considered that these 8 natural food extracts produce marked antimutagenic effects and are practically valuable in the field of chemoprophylaxis of liver cancer in humans.


Comparison of chemically induced chromosome loss in a diploid, triploid, and tetraploid strain of Saccharomyces cerevisiae.

Triploid and tetraploid strains of Saccharomyces cerevisiae were constructed and the spontaneous loss during mitosis of one, two or three copies of chromosome VII was determined. In one strain, a triploid (VM2) in which expression of the recessive alleles can be observed only after loss of two copies of chromosome VII (3N-2), the spontaneous frequency of chromosome loss was lower than in the diploid D61.M. In another strain, a tetraploid (VM4) that also requires the loss of two copies of chromosome VII for observation (4N-2) of the recessive alleles, the spontaneous frequency was slightly higher than in the diploid D61.M. The spontaneous frequency of other genetic events (that is, mutation, recombination or chromosome breakage) were lower by 2-3 orders of magnitude than in the diploid strain D61.M. Induction of chromosome loss and other genetic events by nocodazole, ethyl acetate, hydroxyurea and ethyl methanesulfonate was determined in D61.M, VM2, and VM4, and the results were compared. Nocodazole and ethyl acetate induced chromosome loss in both the triploid and the tetraploid strains at lower concentrations than required in the diploid. These compounds also induced elevated frequencies of other genetic events in both the triploid and the tetraploid strains but not in the diploid. Hydroxyurea induced elevated frequencies of chromosome loss in the diploid and the tetraploid. Frequencies of chromosome loss in the triploid treated with hydroxyurea, although elevated, are based on observation of very few colonies of the correct phenotype. Ethyl methanesulfonate failed to induce chromosome loss in any of the three strains. Hydroxyurea and ethyl methanesulfonate did, however, induce very high frequencies of other genetic events.


Micronucleus induction and phagocytosis in mammalian cells treated with diesel emission particles.

Micronucleus induction and phagocytosis in V79 and CHO cells treated with diesel emission particles (DEP) were studied. After separation of the sample into supernatant and sediment fractions, the genotoxic activity of DEP was shown to reside in the supernatant fraction for the DMSO-extracted sample, and in the sedimented fraction for the dipalmitoyl lecithin (DPL), a primary component of pulmonary surfactant, dispersed sample. More particles from DMSO sediment samples were phagocytized than DPL sediment by both types of cells. This had no effect, however, on micronucleus induction. CHO cells phagocytized fewer particles, but gave a higher number of micronuclei than V79 cells. CHO cells seem to be more sensitive to DEP. Evidently, micronucleus induction is not the result of phagocytosis per se, but is due to the different response of the indicator cells to the DEP sample tested. These results further indicate that most, if not all, genotoxic compounds associated with DEP can be extracted by DMSO and that genotoxic activity associated with DEP inhaled into the lung may also be expressed by dispersion of particles in pulmonary surfactant.


Mutagenic activity of a series of synthetic and naturally occurring heterocyclic amines in Salmonella.

26 synthetic and naturally occurring heterocyclic amines were tested in the Salmonella/microsome assay (Ames test) using tester strains TA98 and TA100 in the presence of an Aroclor-induced rat-liver S9 fraction. 9 of the compounds were protein-pyrolysis products which had previously been shown to be mutagenic. Mutagenic potencies similar to previously reported values were demonstrated for these compounds with the exception that Trp-P-1 was only mutagenic in strain TA98 in our study, although it had previously been reported to be weakly mutagenic in strain TA100. 17 structurally diverse heterocyclic amines were synthesized and tested for mutagenicity. The structural diversity of these synthetic heterocyclic amines will enhance the sensitivity of future quantitative structure-activity relationship (QSAR) studies by demonstrating the structural characteristics essential for mutagenicity. The results of this study provide a large data base for the mutagenicity of this important class of compounds.


Importance of the type of soil for the induction of micronuclei and the growth of primary roots of Vicia faba treated with the herbicides atrazine, glyphosate and maleic hydrazide.

Research was carried out on the genotoxic effects (induction of micronucleated cells in primary root tips) and toxic effects (reduction in primary root growth) in young plants of Vicia faba grown in soils with different organic matter contents and treated with the herbicides atrazine, glyphosate and maleic hydrazide. The data obtained show that the genotoxic effects are noticeably influenced by the interactions between the herbicide and the type of soil in which the Vicia faba have grown. While maleic hydrazide proved to be highly clastogenic for young plants grown in both soils, atrazine was genotoxic only in young plants grown in soil poor in organic matter. Glyphosate did not induce micronuclei under either soil condition, but induced a significant toxic effect.


[New approaches in speech therapy].

Following a brief introduction of the neurological basis and the localization of aphasias, we discuss approaches to language therapy which are based on linguistic theories. We first sketch principles of language-based therapy. Then we introduce communicative aphasia treatment and explain aspects under which the latter represents an extension of the language-based approach. In order to provide neurological arguments in favour of communicative therapy, we discuss the language theory of Hughlings Jackson. Finally, we give examples for communicative therapy paradigms, for some of which implementation in specific computer training programs is available.


Neurons in the rat medulla oblongata containing neuropeptide Y-, angiotensin II-, or galanin-like immunoreactivity project to the parabrachial nucleus.

Projections from the medulla to the parabrachial complex of the rat were examined for their content of neuropeptide Y-, angiotensin II- or galanin-like immunoreactivity using combined retrograde tracing and immunohistochemical techniques. Rhodamine-labelled latex microspheres were stereotaxically injected into discrete nuclei of the parabrachial complex. After survival of two to five days, colchicine (100 micrograms in 10 microliters saline) was injected into the cisterna magna. One day later, rats were perfused and the brainstems were prepared for visualization of the retrograde tracer and immunoreactivity of one of the three peptides. Retrograde labelling verified that the area postrema, nucleus of the tractus solitarius, caudal spinal nucleus of the trigeminal nerve, parvocellular reticular nucleus, and ventrolateral medulla including the rostral ventrolateral medulla and nucleus paragigantocellularis project to the lateral parabrachial and Kölliker-Fuse nuclei. While most projections were primarily ipsilateral, a small proportion of the projections from the ventrolateral medulla was bilateral. Neurons containing neuropeptide Y-like immunoreactivity were found in the caudal and intermediate nucleus of the tractus solitarius, dorsal to the lateral reticular nucleus and in the nucleus paragigantocellularis. After bilateral microsphere injections into the lateral parabrachial and Kölliker-Fuse nuclei, double-labelled neurons were found dorsal to the lateral reticular nucleus of caudal and intermediate medullary levels, at the ventral surface of the medulla at intermediate levels and in the nucleus paragigantocellularis at rostral levels. Neurons with angiotensin II-like immunoreactivity were observed at the dorsomedial border of the caudal and intermediate nucleus of the tractus solitarius, in the area postrema and in the lateral reticular nucleus and nucleus paragigantocellularis. Of these neurons, small numbers in the nucleus of the tractus solitarius and ventrolateral medulla also projected to the lateral parabrachial and Kölliker-Fuse nuclei. Neurons containing galanin-like immunoreactivity were found in the caudal nucleus of the tractus solitarius, the area postrema, the spinal trigeminal nucleus, the raphe nuclei (pallidus and obscurus), the nucleus paragigantocellularis and dorsal to the lateral reticular nucleus. Of these cells, double-labelled neurons were found in the commissural and medial subdivisions of the caudal nucleus of the tractus solitarius and in the rostral ventrolateral medulla including the ventral surface and the nucleus paragigantocellularis. The results suggest that neuropeptide Y, angiotensin II and galanin may serve as neurochemical messengers in pathways from the medulla to the parabrachial complex. The location of double-labelled neurons suggests that the information relayed by these neurons is related to autonomic activity.


Afferent fibres from the guinea-pig ureter: size and peptide content of the dorsal root ganglion cells of origin.

A study was undertaken to determine the segmental organization of the dorsal root ganglion cells which give rise to ureteric primary afferent fibres in the guinea-pig. The size-distribution and peptide content of these dorsal root ganglion cells were examined and compared with a sample of all dorsal root ganglion cells from the same ganglia. Afferent fibres to the guinea-pig ureter were found to arise mainly from dorsal root ganglia L2-L3 and S1-S2. A large contralateral component of the afferent innervation of the ureter was found when either the right or the left ureter was injected with tracer. This amounted to approximately 40% of the total labelled cells. The cross-sectional areas of the dorsal root ganglion cells of ureteric afferents were found to be at the smaller end of the size-range for the whole ganglion. Most (90%) of the cells innervating the ureter were immunoreactive for one of the peptides studies, substance P or calcitonin gene-related peptide, and a large proportion (65%) were immunoreactive for both. This was very different for the ganglia as a whole, where only about 50% of the cells were immunoreactive for either of the peptides and only 14% were immunoreactive for both peptides. These results show a bilateral afferent innervation of the ureter by nerve fibres which, in the vast majority, contain substance P and/or calcitonin gene-related peptide.


Glial cells from adult rat olfactory bulb: immunocytochemical properties of pure cultures of ensheathing cells.

Three morphologically and immunohistochemically distinct types of cell were present in primary cultures of adult rat olfactory nerve and glomerular layers of the olfactory bulb. One cell type was multipolar and stained positively for glial fibrillary acidic protein; a second type had fried egg-like morphology and stained with antibodies to epitope ED1; the third cell type had fusiform morphology, reacted with antibodies to vimentin and laminin and was glial fibrillary acidic protein- and ED1-negative. Trypsinization of these primary cultures (3 min, 37 degrees C), detached multipolar and fusiform cells only. When detached cells were set up in secondary culture on a glass substrate, fusiform cells did not attach, resulting in a pure culture of multipolar cells. Multipolar cells were glial fibrillary acidic protein- and myelin basic protein-positive and had the properties of so-called ensheathing cells or Blanes' glia. Immunoreactivity with anti-nerve growth factor receptor and anti-fibronectin allowed us to identify four distinct populations of multipolar ensheathing cells. One population was nerve growth factor receptor-positive, fibronectin-negative. A second was nerve growth factor receptor-negative and fibronectin-positive. A third was positive for both markers and the remaining cells did not stain for either of them. The morphological and immunological characteristics of cultured cells from olfactory nerve and glomerular layers were similar to those of Schwann cells and the similarities could account for the permissivity to axonal growth of the olfactory bulb.


Time-dependent neuroplasticity in mesostriatal projections after unilateral removal of vibrissae in the adult rat: compartment-specific effects on horseradish peroxidase transport and cell size.

In adult rats the mystacial vibrissae were clipped on one side of the snout, and the influence of this sensory deprivation on crossed and uncrossed striatal afferents from the substantia nigra, ventral tegmental area, and retrorubral area was examined with the horseradish peroxidase tract tracing technique. Unilateral removal of vibrissae was found to affect crossed and uncrossed nigrostriatal projections in a time-dependent manner. One to three days after hemivibrissotomy an apparent neuronal asymmetry was found in the crossed nigrostriatal projection arising in the rostral part of the substantia nigra, with more labeled neurons in the projection to the caudate-putamen on the side of vibrissae removal. This asymmetry resulted mainly from an asymmetry in the subset of nigrostriatal neurons reported to project to the striatal matrix ("dorsal cell type"). In contrast, 4-20 days after hemivibrissotomy reversed asymmetries were found in crossed and uncrossed nigrostriatal projections, with more labeled neurons in the projections to the caudate-putamen of the hemisphere opposite to vibrissae removal (the sensory deprived hemisphere). The asymmetry in the uncrossed projection was found throughout the substantia nigra, but was also most substantial in the projection from its rostral part. The asymmetry in the crossed projection was again restricted to the rostral substantia nigra; interestingly, however, it was limited to the subset of neurons reported to terminate in the striosomes ("ventral cell type"). Evidence was also found for time-dependent changes in size of neurons of the crossed nigrostriatal projections, as well as for changes in striatal afferents from the ventral tegmental area. The time course of these apparent changes in strength of mesostriatal projections is similar to the known time course of recovery from behavioral asymmetries induced by hemivibrissotomy, which is suggestive of a functional relationship between neuronal and behavioral changes. Moreover, the finding of a differential influence of hemivibrissotomy on nigrostriatal afferents to striosomes and matrix is indicative of a functional dissociation of these two systems.


Alzheimer's disease: is the decrease of the cholinergic innervation of the hippocampus related to intrinsic hippocampal pathology?

Consistent findings in the hippocampi of patients with Alzheimer's disease are the presence of neurofibrillary tangles in pyramidal neurons and the loss of choline acetyltransferase activity due to degeneration of hippocampal cholinergic terminals. The present study sought to clarify, in the brains of five patients with Alzheimer's disease and four controls, whether the loss of cholinergic terminals in the hippocampal stratum pyramidale in Alzheimer's disease is related to degenerative changes in hippocampal pyramidal cells. A polyclonal antibody to human choline acetyltransferase was employed to visualize immunohistochemically cholinergic terminals. Hippocampal neurons were stained with Cresyl Violet, neurofibrillary tangles with thioflavin S and a monoclonal antibody against phosphorylated neurofilament (RT97). Quantification of the stained structures was performed in CA4, CA1 and the subiculum, on five sections selected from the entire anteroposterior extent of each hippocampus. In the group of Alzheimer patients, the densities of cholinergic terminals were homogeneously diminished in the three hippocampal subregions in comparison with the controls (32-33%). In contrast, a significant loss of pyramidal neurons was found only in CA1, and the density of neurofibrillary tangles was markedly increased only in CA1 and the subiculum in Alzheimer's disease. These findings suggest that there is no relationship between the loss of cholinergic terminals and the degeneration of pyramidal cells in the hippocampus of patients with Alzheimer's disease.


Laterality in the vestibulo-cerebellar mossy fiber projection to flocculus and caudal vermis in the rabbit: a retrograde fluorescent double-labeling study.

The vestibular nuclear input into the flocculus and the uvula and nodulus of the caudal vermis was studied in rabbits by means of retrograde transport of the fluorescent tracers Fast Blue and Diamidino Yellow. Through simultaneous injection of the tracers in the homonymous lobules at either side of the midline, the distribution, the preference in laterality and the degree of collateralization of vestibulo-cerebellar neurons could be studied. The nucleus prepositus hypoglossi was included in the analysis. In the nucleus prepositus hypoglossi and in the medial vestibular nucleus 6% of the number of labeled neurons contained both tracers, against 4% in the descending vestibular nucleus. In the superior vestibular nucleus a statistically significant difference in the proportion of double-labeled neurons was found between cases with injections in the flocculus (1%) and the caudal vermis (9%). The relative distribution of single-labeled neurons projecting to either the flocculus or the caudal vermis was similar in most of the vestibular nuclei. A statistically significant preference for a projection to the flocculus in favor of one to the caudal vermis, was found for neurons in the medial vestibular nucleus and the prepositus hypoglossal nucleus. Statistically significant laterality preferences were found in the superior vestibular nucleus for the contralateral flocculus.


Influence of extracellular and intracellular pH on GABA-gated chloride conductance in crayfish muscle fibres.

The effect of intracellular and extracellular pH on GABA-gated Cl- conductance was studied using H(+)-selective microelectrodes and a three-microelectrode voltage clamp in crayfish leg opener muscle fibres in bicarbonate-free solutions. Experimental variation of intracellular pH in the range 6.4-8.0 did not affect the GABA-gated conductance. In contrast to this, the GABA-gated conductance was sensitive to changes in external pH. Raising the external pH from 7.4 to 8.4 decreased the GABA-gated peak conductance observed immediately following application of GABA by 30%, and a change from 7.4 to 6.4 produced an increase of 26%. The effect of extracellular pH on the GABA-gated peak conductance was approximately linear in the pH range 6.4-8.9. A slight decrease in the slope of the pH-conductance relationship was evident in the pH range 5.4-6.4. The desensitization of the GABA-gated conductance was also affected by external pH. At pH 6.9 the conductance produced by 1 mM GABA showed a desensitization of about 15%, and at pH 8.9 this value was 34%. Raising the external pH in the presence of GABA decreased the GABA-gated peak conductance and increased the fractional desensitization, while lowering the external pH produced opposite effects, and was capable of repriming the conductance from a desensitized state to the non-desensitized state. The above results show that the GABA-gated conductance is sensitive to changes in external pH in the physiological range, and suggest that pH-dependent changes in the postsynaptic efficacy of GABA-mediated inhibition may contribute to H+ modulation of neuronal excitability.


Serotonin-induced stimulation of cortisol secretion from human adrenocortical tissue is mediated through activation of a serotonin4 receptor subtype.

The occurrence of serotonin in the human adrenal gland was demonstrated both by immuno-histochemical and biochemical approaches. Using specific polyclonal antibodies to serotonin, the presence of numerous immunoreactive cells was revealed by means of the peroxidase-antiperoxidase technique. These cells exhibited the morphological characteristics of mast cells. Combination of high performance liquid chromatography and electrochemical detection showed the presence of substantial amounts of both serotonin and its metabolite 5-hydroxyindolacetic acid in adrenocortical extracts. The role of serotonin in the regulation of steroidogenesis from human adrenocortical slices was studied in vitro using a perifusion system technique coupled to a specific radioimmunoassay for cortisol. Graded doses of serotonin (from 10(-8) M to 3 x 10(-7) M) increased cortisol production in a dose-dependent manner. Prolonged exposure of adrenal fragments to serotonin (10(-7) M) induced a biphasic response, i.e. a rapid and transient increase in cortisol secretion followed by a plateau phase, suggesting the existence of a desensitization phenomenon. The stimulatory effect of serotonin (10(-7) M) was not altered during infusion of the serotonin1 and/or serotonin2 receptor antagonists methysergide (10(-6) M) and ketanserin (10(-6) M), respectively. In contrast, ICS 205 930 (10(-6) M), a non-selective serotonin3/serotonin4 antagonist, totally abolished the response of adrenal slices to serotonin (10(-7) M). The benzamide derivative zacopride, considered as a serotonin4 agonist, induced a robust stimulation of cortisol secretion. In addition, the corticotropic effects of serotonin (10(-7) M) and zacopride (10(-6) M) were not additive. Incubation of adrenocortical fragments with zacopride (10(-6) M) or serotonin (10(-6) M) caused a significant increase in cAMP formation. Taken together, these data suggest that serotonin, locally released by intra-adrenal mast-like cells, may act as a paracrine factor to stimulate cortisol secretion in man. Our results also indicate that serotonin-induced corticosteroid production is mediated through activation of a serotonin4 receptor subtype positively coupled to adenylate cyclase.


Striosomal organization of substance P-like immunoreactivity in parkinsonian patients.

We designed this study to evaluate the expression of substance P (SP) in the striatum and the globus pallidus in normal controls, idiopathic Parkinson's disease (PD), and striatonigral degeneration (SND). We determined immunohistochemically the expression of SP in formalin-fixed, paraffin-embedded brain tissues using the free-floating method. In normal controls, we observed SP-like immunoreactivity in the internal segment of the globus pallidus, where the SP-positive fibers showed typical "woolly fiber" patterns. SP expression in the striatum had the characteristic patchwork of striosomes. Patients with PD showed the normal distribution pattern of SP. By comparison, in patients with SND, there was a striking diminution of SP in the putamen, while the SP-positive patches persisted in the caudate nucleus. This study represents the first unequivocal immunohistochemical demonstration of SP-positive striosomes in the human striatum.


Effect of clinical status and treatment on the frequency of CD5+ B cells in patients with myasthenia gravis.

CD5+ B cells comprise a subset of B lymphocytes that may play a pathogenetic role in the development of human autoimmune disease. We previously reported a high-frequency phenotype (greater than 30% CD5+ B cells) in the peripheral blood of 57% of myasthenia gravis (MG) patients and 13% of controls. We have now examined additional patients (n = 41) and controls (n = 27) and continue to find that 44% of MG patients have a high-frequency phenotype of CD5+ B cells compared with 7% of controls. Serial studies showed that the frequency of CD5+ B cells in peripheral blood remained fairly stable for controls and that it may decrease with immunosuppressive therapy of MG patients. In patients receiving anticholinesterase only or no therapy (n = 21), the age at onset of MG, presence or absence of detectable serum anti-acetylcholine receptor antibody, clinical extent of MG, and the duration of disease may affect the frequency of CD5+ B cells in the blood.


Levels of CD5+ B lymphocytes do not differ between patients with myasthenia gravis and healthy individuals.

CD5+ B cells might be involved in autoimmunity mainly as autoantibody-producing cells. To investigate the possible role of these cells in myasthenia gravis, we studied the numbers of CD5+ B cells, CD5- B cells, and CD19+ B cells as well as CD5+ T cells in the peripheral blood from 31 patients with myasthenia gravis and 31 healthy individuals. Both absolute percentages (percent of peripheral blood mononuclear cells) and relative percentages (percent of total CD19+ B cells) of CD5+ B cells were the same in patients as in controls. The numbers of CD5- B cells and CD19+ B cells were the same in both groups, whereas CD5+ T cells were lower in the patients. There was no correlation between clinical stage, sex, thymectomy, or pathology of thymus and the levels of CD5+ B cells, CD5- B cells, or CD19+ B cells. Patients treated with azathioprine had lower levels of CD5+ B cells than untreated patients and controls. Our results show that patients with myasthenia gravis have the same levels of CD5+ B cells as healthy individuals.


CD4+ T-epitope repertoire on the human acetylcholine receptor alpha subunit in severe myasthenia gravis: a study with synthetic peptides.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) seems crucial in the pathogenesis of the autoimmune paralysis myasthenia gravis (MG) because it contains both the epitopes that dominate the antibody response against the AChR and those recognized by CD4+ AChR-specific T helper (Th) cells. To define the repertoire of anti-AChR Th cells, we investigated the response of unselected blood CD4+ cells or total lymphocytes, or both, from 22 MG patients to 20-residue overlapping synthetic peptides, screening the complete sequence of human-muscle AChR alpha subunit. Several epitopes were identified. Only the most severely affected patients recognized alpha subunit epitopes, and they were mainly young women. Detection of in vitro AChR-specific CD4+ response was facilitated by removal of the CD8+ cells because in two patients a clear response to several alpha subunit peptide sequences could be detected when CD(8+)-depleted cells were used, while their total peripheral blood mononuclear cell population did not respond to any alpha subunit peptide. Although each patient had a unique pattern of peptide recognition, four immunodominant regions recognized by long-term AChR-specific CD4+ T-cell lines, or flanking peptide sequences, were recognized most frequently (residues 48-67, 101-137, 293-337, and 308-437).


[Acquired epileptic aphasia--Landau-Kleffner syndrome].

Authors report about a girl with Landau-Kleffner syndrome. After the history of normal developmental milestones at the age of five-and-half year the patient had absence seizures, and marked receptiv and expressiv aphasia developed gradually. She was found to have Landau-Kleffner syndrome. This entity initially can be regarded as deafness or psychiatric reaction. The seizure activity usually has a favorable outcome, the aphasia can fluctuate for years. The improvement is more likely in promptly treated cases.


Immunohistochemical analysis of synovial membranes from inflammatory and non-inflammatory arthritides: scarcity of CD5 positive B cells and IL2 receptor bearing T cells.

Rheumatoid Arthritis (RA) synovial membranes were examined by single and dual immunohistological techniques with a number of monoclonal antibodies against lymphocyte and macrophage related antigens. CD4 positive T lymphocytes frequently expressed MHC Class II antigens and were found in sublining collections in close association with activated macrophages as well as B lymphocytes. CD8 positive T cells surrounded these collections as well as being scattered throughout the membrane and also frequently expressed MHC Class II antigens. IL2 receptor (IL2r) expression on T cells and CD5 expression on B cells were rarely seen in these synovial membranes. Similar immunohistological architecture was found in synovial membranes from patients with psoriatic arthritis (PA) and Reiter's Syndrome (RS). Normal synovium contained few T cells, with few cells expressing MHC Class II antigens. Synovium from osteoarthritis (OA) patients also demonstrated similar immunohistological changes to those found in inflammatory arthritides, suggesting that there are only quantitative rather than qualitative differences between the synovial membrane immunohistological architecture from patients with inflammatory and noninflammatory arthritides.


Clear cell sarcoma of kidney: report of the first Malaysian case.

Clear cell sarcoma of kidney (CCSK) is a rare but distinct tumor of childhood frequently confused with Wilms' tumor (nephroblastoma). It has a characteristic histology, a marked predilection for metastasis to bone, and an aggressive clinical course with a high relapse rate in spite of surgical excision, chemotherapy and radiotherapy. We report the first histologically proven CCSK in a Malaysian patient. This was an 8-mth-old Malay boy who was clinically diagnosed to have stage I Wilms' tumor. Despite treatment, he developed multiple metastases 10 mths after initial presentation and died soon after. Emphasis is placed on recognizing this entity in view of (1) its naturally aggressive behaviour and (2) the prospect of improving prognosis with currently recommended intensified chemotherapeutic regimes. Its immunohistochemical profile of vimentin-positivity and negativity for epithelial membrane antigen, cytokeratin and Factor-8 related antigen is more in favour of a mesenchymal or glomerular origin than a tubular or vascular origin.


Velo-cardio-facial syndrome associated with ventricular septal defect, pulmonary atresia, and hypoplastic pulmonary arteries.

We report on 15 patients with velo-cardiofacial syndrome who had a severe form of tetralogy of Fallot (pulmonary atresia, ventricular septal defect, and hypoplastic pulmonary arteries). Noncardiac anomalies in these patients included typical facial and ear anomalies in 15, nasal speech in 13, palate anomalies in 10, and developmental delay in 10. Seven patients had significant bronchospasm, which has not been reported in association with the velo-cardio-facial syndrome. All 15 patients had severe abnormalities of the arborization of the pulmonary arterial tree, which also has not been reported in velo-cardio-facial syndrome. All patients underwent staging operations to prepare the true pulmonary vascular tree for complete repair of the defect (five underwent complete repair and three survived). Of the remaining 10 patients, 6 are awaiting further operation, 3 are not candidates for complete repair, and 1 has died.


Reverse transcriptase inhibits Taq polymerase activity.

Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.


Mutations in E.coli 16s rRNA that enhance and decrease the activity of a suppressor tRNA.

The in vivo expression of mutations constructed within helix 34 of 16S rRNA has been examined together with a nonsense tRNA suppressor for their action at stop codons. The data revealed two novel results: in contrast to previous findings, some of the rRNA mutations affected suppression at UAA and UAG nonsense codons. Secondly, both an increase and a decrease in the efficiency of the suppressor tRNA were induced by the mutations. This is the first report that rRNA mutations decreased the efficiency of a suppressor tRNA. The data are interpreted as there being competition between the two release factors (RF-1 and RF-2) for an overlapping domain and that helix 34 influences this interaction.


The topography of the 3'-terminal region of Escherichia coli 16S ribosomal RNA; an intra-RNA cross-linking study.

30S ribosomal subunits, 70S ribosomes or polysomes from E. coli were subjected to mild ultraviolet irradiation, and the 3'-terminal region of the 16S RNA was excised by 'addressed cleavage' using ribonuclease H in the presence of suitable complementary oligodeoxynucleotides. RNA fragments from this region containing intra-RNA cross-links were separated by two-dimensional gel electrophoresis and the cross-link sites identified by our standard procedures. Five new cross-links were found in the 30S subunit, which were localized at positions 1393-1401 linked to 1531-1532, 1393-1401 linked to 1506, 1393-1401 to 1502-1504, 1402-1403 to 1498-1501, and 1432 to 1465-69, respectively. In 70S ribosomes or polysomes the first four of these were absent, but instead two cross-links between the 1400-region and tRNA were observed. These results are discussed in the context of the tertiary folding of the 3'-terminal region of the 16S RNA and its known functional significance as part of the ribosomal decoding centre.


Molecular design of a eukaryotic messenger RNA and its chemical synthesis.

A designed mRNA consisting of 42 ribonucleotides having the cap structure was synthesized. The capped leader sequence of the brome mosaic virus (BMV) mRNA 4, m7G5'pppGUAUUAAUA (F-1), was synthesized by the phosphotriester method and followed by the capping reaction. A 32-mer consisting of an initiation codon (AUG), the coding region corresponding to a bacterial pheromone cAD1 and two stop codons, was constructed by the 18-mer (F-2) and 14-mer (F-3), which were synthesized by the phosphoramidite method. 2'-,3'-O-Methoxymethylene-guanosine 5'-phosphate was condensed with F-3 using P1-2',3'-O-methoxymethyleneguanosine-5'-yl P2-adenosine-5'-yl pyrophosphate (9) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated successively with T4 RNa ligase to afford the whole RNA molecule.


Transformation of a novel direct-repeat repressor element into a promoter and enhancer by multimerisation.

Studies on the regulation of interferon (IFN) responsive genes have mainly been centred on the highly conserved IFN stimulated responsive elements (ISREs) which can mediate type I and II IFN inducibility. To date little is known about other functional cis-acting regulatory motifs in IFN responsive genes. We report here on the identification of a repressor element in the human MxA gene defined to a 19 base pair (bp) region which houses a 9 bp direct repeat. DNA-specific protein binding on this element is not affected by IFN treatment and is distinct from ISRE binding proteins. Remarkably, contrary to expectations, when the repressor element is multimerised and spliced, in either orientation, to a reporter gene it behaves like a functional, constitutive promoter. Positioning the multimerised element in front of the SV40 enhancerless promoter also led to enhanced expression. The same protein(s) seem to bind to both the single repressor element and its multimerised form. This discovery of phenotypic reversal on a repressor element via multimerisation may have important implications in vivo.


The nature of preferred hairpin structures in 16S-like rRNA variable regions.

Variable length hairpins in 16S-like rRNA show a predominance for tetra-loops, its degree correlates with the protein content of the ribosome. The number of base-pairs adjacent to the loop (the tip size) and the nearest neighbor composition contribute to the stability of hairpin structures. The average tip size in length variable hairpins correlates with the thermophilicity of the organism, i.e. in temperate environments less stable stem structures are tolerated or even necessary. The most abundant loop families UUCG, GCAA, and CUUG occur most frequently at loop sizes 3, 2, and 7, respectively. Short tips of size less than or equal to 4 generally prefer nearest-neighbor combinations that result in CCC-GGG. Loop-specific tipmost nearest neighbors are revealed at longer tips: CUC(UUCG)GAG, GUA(GCAA)UAC with a maximum at tip sizes 5-6, and GWG(CUUG)CWC. Conserved hairpins, however, prefer variants of the UUCG and GCAA motifs with additional purines. Minor loop families and single motifs such as UUUA, UUUU, CUUGU, UUCGG, and UUU are investigated for preferable tip sizes and nearest-neighbor composition. Specific features are revealed for prominent hexa-loops.


Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes.

We report here the first successful generation of a chromosome-enriched library from flow sorted plant chromosomes. Chromosomes with a characteristic DNA content (a peak) were sorted from a synchronized cell culture (TaKB1, derived from Triticum aestivum). A HpaII library was constructed from the sorted chromosomes and half of the cloned DNA sequences analysed are unique or low copy. Approximately half of these sequences when used as probes detect sequences on wheat chromosome 4A. The generation and analysis of the chromosome library is described in detail and the prospects of using flow-sorted plant chromosomes discussed.


[Calcofluor-white staining in the identification of Pneumocystis carinii].

Calcofluor white stain is widely used as a fluorescent stain in mycology. Recently, it was found that by calcofluor white staining Pneumocystis carinii cysts exert a highly characteristic staining pattern. We retested fifteen clinical specimens which were known to be positive for Pneumocystis carinii (diagnosis made by a methenamine silver stain). Calcofluor white stain proved to be a simple, rapid, and inexpensive method for detecting Pneumocystis carinii in clinical specimens.


Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.


Regulation of fracture repair by growth factors.

Fractured bones heal by a cascade of cellular events in which mesenchymal cells respond to unknown regulators by proliferating, differentiating, and synthesizing extracellular matrix. Current concepts suggest that growth factors may regulate different steps in this cascade (10). Recent studies suggest regulatory roles for PDGF, aFGF, bFGF, and TGF-beta in the initiation and the development of the fracture callus. Fracture healing begins immediately following injury, when growth factors, including TGF-beta 1 and PDGF, are released into the fracture hematoma by platelets and inflammatory cells. TGF-beta 1 and FGF are synthesized by osteoblasts and chondrocytes throughout the healing process. TGF-beta 1 and PDGF appear to have an influence on the initiation of fracture repair and the formation of cartilage and intramembranous bone in the initiation of callus formation. Acidic FGF is synthesized by chondrocytes, chondrocyte precursors, and macrophages. It appears to stimulate the proliferation of immature chondrocytes or precursors, and indirectly regulates chondrocyte maturation and the expression of the cartilage matrix. Presumably, growth factors in the callus at later times regulate additional steps in repair of the bone after fracture. These studies suggest that growth factors are central regulators of cellular proliferation, differentiation, and extracellular matrix synthesis during fracture repair. Abnormal growth factor expression has been implicated as causing impaired or abnormal healing in other tissues, suggesting that altered growth factor expression also may be responsible for abnormal or delayed fracture repair. As a complete understanding of fracture-healing regulation evolves, we expect new insights into the etiology of abnormal or delayed fracture healing, and possibly new therapies for these difficult clinical problems.


Tumor necrosis factor, interleukin, and interferon induced changes in lipid metabolism as part of host defense.

As the immune response is activated during infection, multiple changes in lipid metabolism, especially increased production of VLDL, occur. Many of the cytokines that mediate the immune response are able to produce such changes in lipid metabolism in vivo. The induction of hypertriglyceridemia or other changes in lipid metabolism during infection do not directly cause the wasting syndrome. It appears that such changes in lipid metabolism may be beneficial to the host, as lipoproteins inactivate a variety of infectious agents. Cytokine-driven hepatic VLDL production during infection most likely represents a part of the acute phase response. The body is thus able to increase serum lipids during infection, or at least maintain triglyceride-rich lipoproteins despite the anorexia of infection. In this manner, the anti-infective, protective effects of lipoproteins are maintained.


Reduced gastric acid inhibitory effect of a pGIP(1-30)NH2 fragment with potent pancreatic amylase inhibitory activity.

Gastric inhibitory polypeptide (GIP) strongly stimulates insulin secretion in the presence of glucose and also stimulates somatostatin release from gastric mucosa. It was reported recently that both stimulatory activities can be dissociated by removing the C-terminal 12 amino acid residues. Since insulin and somatostatin are involved in regulation of exocrine pancreatic and gastric secretion in rats, we compared the inhibitory effects of pGIP and the pGIP(1-30)NH2 fragment on pancreatic amylase and gastric acid secretion. pGIP(1-30)NH2 displayed full activity on inhibition of bombesin (BN)-stimulated amylase release relative to GIP itself, but was about 10-fold less potent in inhibiting gastric acid secretion. These results suggest that the receptors involved in these two events have quite different ligand binding requirements and that more specific analogues of GIP can be designed which should be of value in elucidating the physiological roles of this hormone.


Parasympathetic NANC-secretion of saliva in the mink, and effects of substance P and VIP.

In both parotid and submandibular glands a parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked secretion of saliva was demonstrated. Saliva evoked by exogenous substance P was poor in protein, while saliva evoked by VIP was protein-rich. In a subthreshold dose for fluid secretion VIP released protein and potentiated the responses elicited by substance P, particularly regarding the output of protein. The two neuropeptides may contribute to the parasympathetic NANC secretion of saliva in the mink. Further, agonists responsible for the secretory NANC response are also likely to contribute to the secretory response of the glands to parasympathetic stimulation in the absence of autonomic receptor blockade in this species.


[The immunological characterization of 63 cases of Hodgkin's disease with a panel of 8 antibodies].

Sixty three cases of Hodgkin's disease are studied (two with lymphonodular predominance, 15 with diffuse lymphocyte predominance, 26 nodular sclerosis, 15 mixed cell and 5 lymphocyte depletion) with a panel of 8 monoclonal antibodies, material routinely used and included in paraffin: Ber H2 (CD30), Leu M1 (CD15), Common Leukocyte Antigen (CD45), L26 (CD20), MB2, UCHL1 (CD45 RO), MTI (CD43) and Epithelial Membrane Antigen. Ber H2 turned out to be the most usefull marker, positive in 100% of cases, independently of the histologic type. Positiveness with Leu M1 ranged from 100% (2/2 cases) of lymphonodular predominance, to 53.3% (8/15 cases) of diffuse lymphocyte predominance. The reactivity of the rest was variable, 1 though it is note worthy the positiveness of B markers (L26, MB2) in the two cases with lymphonodular predominance. In the other subtypes, reactivity with L26 was greater than that with MB2. T cell marker expression was minimal, except for the positiveness in 40% of cases (2/5) of the lymphocyte depletion type. In addition, the results of other series are revised and as a result the possible histogenesis of Hodgkin's disease is discussed.


[Current tuberculosis mortality world-wide].

The mortality rate still is an important index for assessment of tuberculosis. Statistical records are kept on the mortality rate on a worldwide basis--more than in the case of other tuberculosis parameters. They allow us to make valuable comparisons. They are also useful because the mortality is closely related to the morbidity. The present thesis is based on comparative figures from the 1989 volume of the WHO Health Statistics Annual. Various countries have been specially selected by the publisher--and subsequently also by us--for sake of clarity. The figures vary strongly within these countries, which was to be expected. The mortality rate varies in Europe (for each 100,000 residents) e.g. from 0.2 in the Netherlands to 8.15 in the Soviet Union. In the Americas the rates vary from 0.4 for Canada to 12.9 for Ecuador. In the Western Pacific region the mortality rates vary from 0.35 for Australia to 14.65 for China. On a worldwide basis, the share of deaths from tuberculosis among all causes of death varies from 0.02% in the Netherlands to 2.10% in the Republic of Korea. The relation of tuberculosis deaths with regard to sexes in Switzerland: 75.7% men, 24.3% women, which is more or less the European average. The lower the mortality rate for tuberculosis are, the lower the difference between the sexes appears to be. Similar facts are found with regard to the distribution of tuberculosis deaths according to age groups: the lower the tuberculosis rate, the more tuberculosis is found in older age groups. The tuberculosis deaths are percentage-wise similarly distributed to the respiratory organs and the other tuberculosis forms.(ABSTRACT TRUNCATED AT 250 WORDS)


The roles of histamine, leukotriene C4 and bradykinin on nasal vascular permeability in experimental nasal allergy of guinea pigs.

The releases of histamine, leukotriene C4 (LTC4) and bradykinin into the nasal cavity were measured following nasal antigenic challenge in ovalbumin (OA) sensitized guinea pigs, or following nasal stimulation with one of these chemical mediators in OA-non-sensitized animals. In sensitized animals, increased vascular permeability of nasal mucosa was recognized immediately after antigenic stimulation and lasted for 90 minutes. The release of histamine into the nasal lavage fluid was observed only immediately after the antigenic stimulation. The releases of LTC4 and kinins into the nasal lavage fluid were augmented not only immediately after the antigenic challenge, but also 60 to 90 minutes after the stimulation. Nasal stimulation with one of these chemical mediators also increased nasal vascular permeability, but lasted for less than 40 minutes. These results suggest that the antigen-induced release of these chemical mediators might play some important roles in early increase of nasal vascular permeability, and that the increase of LTC4 and kinin levels might be involved in the prolonged nasal vascular permeability after nasal allergic response.


Multiple ICAM-1 (CD54) epitopes are involved in homotypic B-cell adhesion.

Two monoclonal antibodies (MoAbs), F10.2 and F10.3, were selected for their ability to interfere in homotypic adhesion of human B cells. Precipitation studies and binding to intercellular adhesion molecule 1 (ICAM-1, CD54) cDNA transfected COS cells revealed that both MoAbs are directed against ICAM-1. The binding of MoAb F10.2 was inhibited by LB-2, a MoAb recognizing the NH2-terminal immunoglobulin-like domain of ICAM-1. This suggests that the epitope recognized by F10.2 is located on the first domain of the ICAM-1 molecule. Binding of the other MoAb, F10.3, was not inhibited by F10.2 nor by two other MoAbs mapping to the first domain of the ICAM-1 molecule. The ability of F10.3 to bind to ICAM-1 is influenced by glycosylation, suggesting that this epitope is located on one of the domains carrying possible glycosylation sites, i.e. domain 2, 3 or 4. The ICAM-1 epitopes recognized by F10.3 and LB-2 or F10.2 co-operated in homotypic adhesion of cells from the EBV cell line ML1. These results suggest that in addition to an epitope located on domain 1 of the ICAM-1 molecule, another epitope whose exposure can be regulated by glycosylation is involved in homotypic B-cell adhesion of cell line ML1.


Expression of CD26 (dipeptidyl peptidase IV) on memory and naive T lymphocytes.

It has been suggested that human CD26-positive T lymphocytes represent the memory pool of the cellular immune system. For proof of this suggestion we analysed the responsiveness of CD26-positive and CD26-negative T lymphocytes after antigenic stimulation in limiting dilution experiments. After stimulation with tetanus toxoid (TT) the number of proliferating cells within the CD26-positive subset was two- to sixfold higher than that within the CD26-negative subset. These differences in responsiveness were also detectable between CD4+/CD26+ and CD4+/CD26- T cells. To further investigate the memory character of the cells, human peripheral blood mononuclear cells were stimulated with TT for 7 or 14 days. Thereafter, CD26+ and CD26- T cells were isolated and restimulated with TT in limiting dilution experiments. Responding cells were found not only within the CD26-positive subset but also within the CD26-negative subset, and their number increased with time. Surface marker analysis of freshly isolated human T lymphocytes or T-cell clones indicated that CD26 is not a stable cell surface marker. Furthermore, CD26 is both absent and present on CD29-positive or CD45RA-positive cells, indicating no association of CD26 with surface markers for memory or naive T cells, respectively. These results strongly argue against the hypothesis that CD26-positive T cells represent the memory pool. We conclude that CD26 is an activation marker of T lymphocytes, which is associated with reactivity on naive and memory cells.


[Has the care of dying patients in hospitals changed? A study of terminal care at the Diaconal hospital in 1977 and 1987].

A research project conducted at the Norwegian Lutheran Hospital in 1977 (213 deaths) was repeated in 1987 (100 deaths). The purpose was to discover if and how terminal care had changed during the ten years. Data sources were case records, nurses' reports, interviews with closest relatives about 12 weeks after the death (response 95% in 1977 and 72% in 1987). Openness about death had not increased. There was some improvement of palliative treatment. In 1987, 79% of all terminal patients had received such treatment, compared with 51% in 1977. The percentage receiving life prolonging treatment, such as parenteral supply of fluids, had fallen from 62% in 1977 to 25% in 1987, and the percentage treated with antibiotics from 31% in 1977 to 15% ten years later.


[Accidents with radioactive pollution. Can we do something?].

Nuclear accidents are a cause of widespread fear. Treatment of patients exposed to high doses of total body irradiation has very often been ineffective. The use of hematopoietic growth factors may be a new way of treating these patients. This article describes results from studies of total body irradiated animals and clinical experience from treating humans who have been involved in nuclear accidents. G-CSF or GM-CSF administered within three hours after a nuclear accident seems to be of value. Surprisingly, so far bone marrow transplantation seems to be of limited value. Intravenous transfusions of thrombocytes and red blood cells, and the treatment of burns and gastrointestinal symptoms, are important parts of the medical therapy.


Mechanism of detection of acute cerebral ischemia in rats by diffusion-weighted magnetic resonance microscopy.

The aim of this study was to measure apparent diffusion coefficients in rat brain tissue exposed to ouabain, glutamate, and N-methyl-D-aspartate and to compare them with apparent diffusion coefficients found in acute cerebral ischemia. The apparent diffusion coefficient was measured using magnetic resonance microscopy in four groups of Sprague-Dawley rats after occlusion of the right middle cerebral artery and ipsilateral common carotid artery (n = 7), after ouabain exposure (n = 6), during glutamate exposure (n = 7), or during N-methyl-D-aspartate exposure (n = 3). Ouabain, glutamate, and N-methyl-D-aspartate were applied via an intracerebrally implanted microdialysis membrane. Three hours after the induction of focal cerebral ischemia, a 33% reduction in the apparent diffusion coefficient was observed in the right dorsolateral corpus striatum and olfactory cortex. After ouabain exposure, reductions in the apparent diffusion coefficient were observed within a 1,500-microns radius of the microdialysis membrane. Quantitative analysis revealed that apparent diffusion coefficient values in ischemic and ouabain-exposed tissue fell within the same range. Glutamate and N-methyl-D-aspartate reduced the brain tissue apparent diffusion coefficient by 35% and 40%, respectively. On the basis of these findings, we conclude that ischemia-induced apparent diffusion coefficient reductions are likely caused by a shift of extracellular to intracellular water.


Serum prostate-specific antigen after post-prostatectomy radiotherapy.

External beam radiotherapy was administered to 39 patients after radical prostatectomy for adenocarcinoma. Thirty-seven of 39 patients had detectable levels of serum prostate-specific antigen (PSA) prior to irradiation as evidence of residual carcinoma (biochemical evidence of disease). Two patients also had palpable recurrences. Pathologic analysis of the surgical specimens suggested that positive surgical margins, seminal vesicle or lymph node involvement, or high Gleason pattern scores are associated with measurable PSA after surgery. Follow-up ranged from two to seventy-four months (mean 26.8 months). To date, local control has been achieved in all but 1 patient (including 2 patients with palpable tumor prior to radiotherapy). Two distinct risk groups for the development of distant metastases based on the trend of the PSA in relation to the duration of follow-up after radiotherapy are defined. In the high-risk group (those patients with a rising PSA), in 9 of the 18 bone metastases have developed, while none of the 17 low-risk patients have metastatic disease.


High-temperature short-time heat inactivation of HIV and other viruses in human blood plasma.

An ultra-short-time heating system was used to process blood plasma spiked with various viruses (HIV, vesicular stomatitis virus, encephalomyocarditis virus). Virus reduction and recovery of plasma proteins were measured at various temperatures from 65 to 85 degrees C. Processing at 77 degrees C and 0.006 s resulted in a high level of virus kill, including greater than or equal to 4.4 log10 HIV, while maintaining protein structure and activity essentially intact.


Improved serodiagnosis of posttransfusion hepatitis C virus infection by a second-generation immunoassay based on multiple recombinant antigens.

Serial serum samples from 35 patients with posttransfusion non-A, non-B hepatitis in a prospective study were tested for antibody to hepatitis C virus (HCV) by a multiple recombinant antigen based immunoassay [anti-HCV (2nd); Abbott]. Of them, 23 were positive for anti-C100, and 27 were positive for HCV RNA by polymerase chain reaction. By anti-HCV (2nd), 28 patients were positive, and all except 1 of the 28 patients were positive for HCV RNA. A total of 24 patients, who became HCV RNA positive at the acute stage and who were negative for both anti-C100 and anti-HCV (2nd) before transfusion, were considered to have posttransfusion hepatitis C. The mean time to seroconversion for anti-HCV (2nd) was 7.5 or 12.1 weeks after the onset of hepatitis or the date of transfusion, respectively, and was generally 6 weeks earlier than that detected by anti-C100. However, seroconversion was delayed in 2 hepatitis B surface antigen carriers as compared with anti-C100. The anti-C100 assay detected 20 (83%) and the anti-HCV (2nd) all 24 patients with documented posttransfusion hepatitis C. The second-generation test is, therefore, better than conventional anti-C100 for the early diagnosis of HCV infection.


Autoantibodies in HIV-infected hemophilia patients against different epitopes on CD4+ lymphocytes and recombinant CD4.

We studied 684 sera obtained from 20 hemophilia patients with AIDS/AIDS-related complex (ARC), 89 asymptomatic HIV+, 76 HIV- hemophilia patients and 151 healthy controls for antibodies against recombinant CD4 (rCD4). Twenty-two percent of AIDS/ARC patients, 10% of asymptomatic HIV+ patients, 17% of HIV-patients, and 1% of healthy controls had anti-rCD4 antibodies. Purified anti-rCD4 antibodies did not react with human CD4+ lymphocytes. This may explain why formation of anti-rCD4 antibodies correlated neither with the occurrence of autoantibodies against CD4+ lymphocytes nor with a decrease in CD4+ cell counts. Antibodies that were eluted from CD4+ lymphocytes after sequential adsorption and elution with separated CD8+ and CD4+ cells reacted with CD4+ lymphocytes of only some healthy individuals, suggesting diversity of CD4 expression.


Substance P- and calcitonin gene-related peptide-like immunoreactive neurons in the rat trigeminal ganglion--with special reference to meningeal and pineal innervation.

The distribution of perikarya showing substance P- (SP) or calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the rat trigeminal ganglion (TG) were investigated by means of immunohistochemical methods. Approximately 50% of the perikarya contain CGRP while SP-LI was observed in 1/3 of the cells. IR fibres were seen to leave the ganglion via the ophthalmic, maxillary, and mandibular nerves. The combination of peptide histochemistry and retrograde labelling of cells in the ganglion following injection of a fluorescent tracer into the pineal gland reveals that few SP- or CGRP-LI trigeminal neurons innervate the pineal gland. In contrast, the vast majority of perikarya in the TG were labelled upon application of the tracer to the meningeal surface supporting the view that meninges and meningeal arteries in rodents are heavily innervated by SP- and CGRP-LI trigeminal neurons.


Treatment of dogs in hemorrhagic shock by intraosseous infusion of hypertonic saline and dextran.

Under isoflurane anesthesia, 50% of the calculated blood volume was removed from 11 dogs. After 30 minutes, five dogs were treated with hypertonic saline and dextran (HSD) (5 mL/kg) followed by isotonic saline solution (2 mL/kg) intraosseously. Six dogs (controls) received isotonic saline (7 mL/kg) intraosseously. All treatments were administered through the medullary cavity of the tibia over a 30-minute period. Cardiac output, mean arterial pressure, central venous pressure, packed cell volume, total protein, and blood gases were monitored for 4 hours. Cardiac output, mean arterial pressure, and circulating volume (indicated by packed cell volume and total protein) were significantly improved after administration of HSD. We conclude that intraosseous infusion of HSD is efficacious in treating hemorrhagic shock and believe the technique may prove to be useful in clinical situations when intravenous lines cannot be established rapidly.


Penetration of lomefloxacin into human prostatic tissue.

Serum and prostatic tissue concentrations of lomefloxacin were measured in 12 elderly patients who underwent transurethral resection of the prostate after receiving a single oral dose of 400 mg. The peak serum concentration of lomefloxacin was 2.5-10.0 micrograms/mL (mean, 5.2 +/- 1.9 micrograms/mL). During surgery, serum and tissue concentrations averaged 4.6 +/- 2.2 micrograms/mL and 6.5 +/- 2.7 micrograms/g, respectively. The ratio of tissue to serum concentrations was 1.53 +/- 0.54. The levels of lomefloxacin in serum and prostatic tissue were found to be higher than the minimum inhibitory concentration (MIC) values for most urinary tract pathogens.


Case report: alternation therapy with antileukemic agents and recombinant human granulocyte colony-stimulating factor for RAEB in transformation.

Under the assumption that in some patients with refractory anemia with excess of blasts (RAEB), the abnormal clones might be less responsive to granulocyte colony-stimulating factor than normal clones, the authors tried alternation therapy with a recombinant form of this factor (rhG-CSF) and antileukemic agents in the treatment of two patients with RAEB in transformation. After repetition of the short-cycled alternation therapy, the hematologic findings of both patients were completely normalized and have remained so without any adverse side effects under the continuation of this therapy for more than 5 months. Judging from our clinical experience, the alternation therapy may be a new efficient therapeutic strategy for RAEB and some types of slowly progressive leukemia.


Infiltration of helper/inducer T lymphocytes heralds central nervous system damage in human T-cell leukemia virus infection.

Cellular infiltrates in new and old lesions in two cases of human T-cell leukemia virus associated myelopathy (HAM) were analyzed with anti-CD3 antibody and OPD4 antibody recognizing CD4 + CD45RO + T lymphocytes. A subset of CD4 lymphocytes with helper/inducer function and labeled with OPD4 constitutes up to 65% of CD3 cells in new lesions in the pons and the cervical cord. In contrast, nonhelper cells and macrophages were dominant in long-standing spinal cord lesions of these HAM cases and inflammatory lesions in two cases of Japanese encephalitis. Thus, unlike in viral infections, the central nervous system (CNS) tissue damage associated with human T-cell leukemia virus (HTLV-1) infection appeared to be heralded by the infiltration of helper/inducer T cells.


Comparison of an immunoperoxidase "sandwich" staining method and western blot detection of P-glycoprotein in human cell lines and sarcomas.

The applicability of a multilayer immunoperoxidase "sandwich" method (IpS) developed by Chan14 for the amplified detection of P-glycoprotein (Pgp) was investigated. The authors examined 15 formalin-fixed cell lines, as well as formalin-fixed, paraffin-embedded sections from single biopsies of 46 sarcomas. The cell lines included sensitive and multidrug resistant sublines (KB, A2780, MCF-7, HeLa) with various relative degrees of resistance to doxorubicin (Dox). The sarcoma biopsy specimens were selected on the basis of the results obtained in Western blot (WB) detection of Pgp (22 positive and 24 negative by WB) using C219 and C494 monoclonal antibodies to Pgp. The IpS method employed C219. The least resistant cell line in which Pgp could be detected by IpS was fivefold resistant to doxorubicin, whereas Pgp was detected by WB in cells greater than twofold resistant. Cell lines having greater than fivefold resistance to Dox were positive by both IpS and WB analyses. The less resistant cell lines contained more nonreactive cells whereas the highly resistant cell lines showed more homogeneous strong membrane reactions. Among the six cell lines determined to be Pgp negative by WB analysis, no false positive immunostaining by IpS existed. One of 22 WB positive and 7 of 24 WB-negative sarcoma biopsy specimens were positive by IpS methods. Reaction varied and was always focal (a minimum of 3-5 cells, ranging up to 3-4 high power fields) indicating pronounced heterogeneous distribution of Pgp. Thus, WB can detect low average (overall) levels of Pgp in tumor samples but such low concentrations of PgP at the single cell are not detectable by IpS methods. However, IpS can discern among many Pgp-negative cells small subpopulations of immunoreactive cells, which are not detected by WB analysis due to Pgp dilution by the membrane protein of numerous Pgp negative cells. IpS and WB used together as complementary methods can provide more complete information about Pgp distribution and content, particularly in the case of heterogeneous human tumors. The IpS method is more suitable for less drastically treated (not embedded) cell line specimens than for paraffin-embedded (routine) sections. Some modification of the present IpS protocol seems necessary to increase its sensitivity and reduce the disparity with WB results.


Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.


Persistent complement activation on tumor cells in breast cancer.

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer.


Expression of vascular cell adhesion molecule-1 in fibroblastlike synoviocytes after stimulation with tumor necrosis factor.

Rapid expression of mRNA encoding vascular cell adhesion molecule-1 (VCAM-1) was induced by tumor necrosis factor (TNF) in fibroblast-like cells obtained from synovial tissue. Both alternatively spliced forms of VCAM-1 mRNA were detected by polymerase chain reaction in TNF-stimulated fibroblast-like synoviocytes. Western blotting analysis showed that two distinct proteins, reactive with an anti-VCAM-1 anti-sera, were expressed by 2 hours of TNF stimulation in both synoviocytes and human umbilical cord vein endothelial cells (HUVEC). The majority of HUVEC and synoviocytes displayed VCAM-1 surface expression after several hours of TNF stimulation. In contrast, dermal fibroblasts upregulated intercellular adhesion molecule-1 (ICAM-1) but not VCAM-1 expression in response to TNF. These results indicate that VCAM-1 and ICAM-1 expression can be differentially regulated and suggest tissue specific regulation of VCAM-1 expression. Furthermore, these findings may provide an explanation for the chronic retention and activation of long-lived lymphocytes and monocytes, which express VLA-4 (the receptor for VCAM-1), in the synovium in rheumatoid arthritis.


A novel monoclonal antibody to cytokeratin 18 with reactivity toward lung squamous cell carcinomas and adenocarcinomas of various sites.

Antibodies to cytokeratins (CKs) have found extensive application in the differential diagnosis of epithelial tumors. The chain-specific anti-CK reagents appear to be of practical value for further subtyping of carcinomas. The authors have produced a novel anti-CK 18 monoclonal antibody (ACK-156) using a modified immunization procedure that included sequential injections of human epidermal keratin, cyclophosphamide, and enriched cytoskeletal extracts from a human lung carcinoma cell line. This protocol effectively amplified clones with reactivity toward CK epitopes not present in epidermal keratin. Monospecificity of the antibody was confirmed by immunoblot analysis using both total cell lysates and cytoskeletal extracts as antigens. Immunoperoxidase staining of adenocarcinomas from a variety of sites, including lung, was strongly positive. Squamous cell carcinomas of lung were also strongly stained whereas squamous cell carcinomas of head and neck origin were stained focally or not at all. In contrast, several commercially available anti-CK 18 monoclonal antibodies did not distinguish squamous cell carcinomas of lung from those of head and neck origin. Immunoblot analysis of tumor lysates corroborated the tissue staining results and revealed that the commercially available antibodies that were tested recognize at least one other low molecular weight peptide in addition to the CK 18 peptide recognized by ACK-156.


Expression of the bcl-2 gene product in follicular lymphoma.

Expression of bcl-2 protein was analyzed in 140 cases of follicular lymphoma by immunohistologic staining of paraffin-embedded tissue; 85% of cases were positive, the frequency being related to histologic grade (100% for the small-cleaved cell type, 86% for the mixed cell type, and 76% for the large cell group). There was striking heterogeneity of bcl-2 content in a number of cases and the smaller neoplastic cells (i.e., centrocytes) were usually the most strongly labeled. In most cases, bcl-2 protein staining was much weaker in normal lymphoid cells than in the neoplastic cells. In several cases, staining for bcl-2 revealed patterns of neoplastic cell spread into adjacent tissue (e.g., normal follicles, lymphoid sinuses), and bcl-2 protein expression tended to be highest in these migratory cells.


Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage.

The authors have tested the hypothesis that plasma membrane disruptions are an early form of structural damage to the fibers of eccentrically exercised muscle. Rat serum albumin (RSA) was used as a marker for muscle-fiber wounding in the rat tricep (medial head) exercised eccentrically by downhill running. In all muscles examined, strong staining with a horseradish peroxidase (HRP)-conjugated anti-RSA antibody was observed between fibers (intercellular staining) and also within certain fibers (intracellular staining). This intracellular staining was interpreted as identifying muscle fibers wounded at their plasma membranes and hence rendered transiently or permanently permeable to extracellular RSA. The most striking finding of this study was a 6.9-fold increase relative to unexercised controls in the number of wounded cells in the medial head immediately after eccentric exercise. The authors also reproducibly observed, albeit less frequently, myocytes that stained with anti-RSA in the medial head and several other muscles of the "unexercised," caged laboratory rat. The extreme vulnerability of muscle plasma membranes to mechanically induced stress was revealed in this study by HRP injections into the triceps long head. A single injection of 200 microliters HRP through a 26-gauge needle resulted in extensive labeling of the muscle fibers present in the long head cross-sectioned at the injection site. The authors propose that initially resealable and/or highly localized, unsealable membrane wounds are an early form of exercise-induced damage that could progress along the length of the fiber until, 1 to 4 days after eccentric exercise, it becomes sufficiently severe that it can be readily recognized as the frank fiber necrosis and cellular infiltration described in numerous previous studies. In possessing cells wounded at their plasma membranes, normal, undisturbed rat muscle and eccentrically exercised muscle appears to resemble gut and skin, two additional tissues routinely exposed to mechanical forces in vivo. The authors propose that membrane disruptions provide a route into and out of myofiber cytoplasm distinct from the conventional, membrane-bounded routes of endo- and exocytosis, and therefore may be of importance both technically, as a route for introducing foreign genes into muscle cells, and biologically, as a route for release of the growth factor, basic fibroblast growth factor.


DNA ploidy abnormalities in the liver of children with hereditary tyrosinemia type I. Correlation with histopathologic features.

Hereditary tyrosinemia (HT) is an autosomal recessive disorder of tyrosine metabolism that results in cirrhosis and hepatocellular carcinoma early in life, and that may be a useful model of early malignant transformation. This is the first report of DNA ploidy in this disease. The authors studied formalin-fixed liver tissue in three cases (two chronic and one acute) of HT for the presence of DNA aneuploidy by flow cytometric (FCM) analysis and image analysis (IA). In the chronic cases, the authors found DNA aneuploidy by FCM in 2/20 cirrhotic nodules in one case and 1/21 tissue sections in the other. Questionable minor aneuploid peaks were detected by FCM in 2/20 and 2/21 sections in these two cases, respectively. Static DNA measurements by IA were performed on those sections having abnormal histograms as well as in some sections having diploid DNA distribution. By this method, the authors confirmed the results of FCM studies and found additional small aneuploid nodules not detected by FCM, frequently associated with various forms of hepatocellular dysplasia as well as steatosis. In one case of acute HT (autopsy), no definite aneuploid peaks were present in five blocks. By immunohistochemical analysis, the authors found frequent positive staining for alpha-fetoprotein (AFP) in the cirrhotic nodules of the two chronic cases as well as in the steatotic regenerative nodules of the acute case. The expression of AFP may represent disturbed regeneration and maturation of liver cells in this disease. This study shows that DNA ploidy may be a useful marker for early malignant transformation in HT and supports the preneoplastic nature of the hepatocellular dysplasia in this disease.


Early microvascular changes in murine cerebral malaria detected in retinal wholemounts.

Changes in the cerebral microvasculature such as breakdown of the blood-brain barrier, petechial hemorrhages, congestion, and edema are observed in the later stages of murine cerebral malaria. These changes have been described from histologic sections of brain, but the need to section the material makes direct observation of the microvasculature in situ difficult. The retinal vasculature, in contrast, offers a unique opportunity to study rheologic, barrier, and functional properties of the microvasculature as a wholemount preparation with normal spatial relationship with other tissues and as an intact vascular plexus. A combination of techniques, including intravascular perfusion of Evan's Blue, Bisbenzimide and Monastral Blue, and fluorescence and transmitted light observation of retinal wholemounts, were developed to examine the progressive microvascular changes in murine cerebral malaria. These techniques allowed detection of phenomena such as monocyte adherence to endothelial cells, congestion, small hemorrhages, and breakdown of the blood-retinal barrier, with details of the location of this leakage, earlier than was possible by studying brain sections. Because the retina is intact, the phenomena were seen in greater detail and some, such as occlusion of vessel segments, were detectable only in retinal wholemounts. In addition, the covisualization of the blood elements, barrier properties, and vascular endothelial integrity that are possible with retinal wholemounts allowed detailed analysis of the interaction of different cellular elements in the pathogenesis of cerebral malaria. Except for detection of edema, the retinal wholemount technique offers a more powerful and less time-consuming technique for detecting early microvascular changes in murine cerebral malaria. This technique could find wider application in the study of other diseases that affect the microvasculature of the central nervous system, such as experimental allergic encephalitis and meningitis.


Correlates of maternal directiveness with children who are developmentally delayed.

Interactions between 25 mothers and their children with developmental delays were correlated to determine the relationships between maternal directiveness and a) maternal behavior regarded as developmentally facilitative, b) maternal intrusiveness, and c) child developmental competence and behavioral engagement. The findings, many of which challenge common conceptions about the nature and role of maternal directiveness, are discussed in relation to these conceptions and to the potential role of directiveness in the development of children with handicaps.


A shared epitope in the acetylcholine receptor-alpha subunit and fast troponin I of skeletal muscle. Is it important for myasthenia gravis?

The monoclonal antibody MAb 155, isolated by Tzartos et al, recognizes the alpha subunit of acetylcholine receptor (AChR) and stains type II skeletal muscle fibers but does not decorate heart muscle. In addition it reacts with most myasthenia gravis-associated thymomas. The authors show by immunoblotting techniques that the myofibrillar antigen is a 23 kd protein and by partial protein sequence data identify it as fast troponin I. Fast troponin I from various species contains the sequence EEKSGMEGRK close to the C-terminal end at positions 165 to 174. The first lysine (K) is crucial for MAb 155 reactivity since its substitution by methionine and leucine in slow troponin I and cardiac troponin I, respectively, abolishes MAb 155 reactivity. The epitope identified on troponin I is homologous in sequence with the MAb 155 epitope on the AChR alpha subunit established by direct peptide binding as KSAIEGIK (positions 373-380). The authors consider whether fortuitously shared epitopes can be responsible for the high level of autoantibodies to AChR and to muscle proteins seen in many MG patients.


Therapeutic approaches to hemoglobin switching in treatment of hemoglobinopathies.

The past decade has witnessed profound increases in knowledge of the structure, function, and developmental regulation of the human globin genes. This information has deepened our understanding of the molecular and cellular mechanisms underlying inherited disorders affecting hemoglobin, and it has provided a new perspective for attaining meaningful increases in fetal hemoglobin synthesis in the management of sickle cell anemia and beta thalassemia. Efforts to provide therapy for these disorders are based on three factors: an understanding of their pathophysiology; the potential for fetal hemoglobin to alter its manifestation; and the concept that developmental changes in globin gene expression might be reversed by manipulating cellular and molecular regulatory mechanisms. In this review we discuss these topics and examine critically recent efforts to apply various pharmacological agents to in vitro, animal, and human models with the goal of increasing HbF synthesis. Several agents have demonstrated activity in patients with hemoglobin disorders. One such agent, hydroxyurea, has been shown to be potentially efficacious in phase II clinical trials in patients with sickle cell anemia and awaits testing in a placebo-controlled phase III study.


Antithrombins: their potential as antithrombotic agents.

The inhibition of thrombin is the key to the prevention and treatment of thrombotic disorders. Although heparin is an extremely effective anticoagulant, it has certain limitations that are not shared by newer thrombin inhibitors. As a result, these novel inhibitors may have advantages over heparin in certain clinical settings.


Study of multidrug resistance (mdr1) gene in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is one of the tumors that shows intrinsic drug resistance to various chemotherapeutic agents. We investigated a possible role of the multidrug resistance (mdr1) gene amplification using a DNA slot blot analysis in 23 untreated non-small-cell lung cancer tissues and 14 corresponding adjacent normal lung tissue samples. In all instances, whether tumors or adjacent normal tissue samples, DNA amplification was not detected except in 1 adenocarcinoma and 2 squamous cell carcinomas that had a low level of amplification of mdr1 gene. In addition, 6 untreated tumors and 7 normal tissue samples were examined for mdr1 RNA expression using RNA slot blot analysis. Only low levels of mdr1 expression were observed in all cases. We conclude that mechanisms other than mdr1 amplification or expression account for the intrisic drug resistance of non-small-cell lung cancers.


Fetal haemoglobin and early manifestations of homozygous sickle cell disease.

The relevance of fetal haemoglobin (HbF) concentration to the development of early clinical manifestations of homozygous sickle (SS) disease has been investigated by examining the time to first occurrence and the proportional hazard of these complications in three groups of the HbF distribution at age 5 years. HbF was significantly related to dactylitis, painful crises, acute chest syndrome, and acute splenic sequestration. The relationship suggested that a critically low HbF concentration increased the risk, little difference in risk occurring between the medium and high HbF groups. The abdominal painful crisis and hypersplenism were not related to HbF concentration suggesting that the degree of sickling may not be important in their genesis. Parental education on acute splenic sequestration should be focused on children with HbF concentrations in the lowest part of the HbF distribution for age.


Peri- and postoperative changes in serum levels of four tumor markers and three acute phase reactants in benign and malignant gynecological diseases.

Serum levels of squamous cell carcinoma antigen, carcinoembryonic antigen, CA 125, tissue polypeptide antigen, CRP, alpha 1-antitrypsin and haptoglobin were determined peri- and postoperatively in patients undergoing surgery for benign gynecological disease (n = 18) and postoperatively in women operated for cervical carcinoma (n = 23). The only significant changes seen after premedication, during anesthesia and during surgery were a decrease in serum concentrations of alpha 1-antitrypsin and haptoglobin. We found no postoperative changes in the serum levels of squamous cell carcinoma antigen nor in carcinoembryonic antigen values. However, the latter analyte was influenced by smoking habits. Elevated levels of CA 125 and tissue polypeptide antigen were found in the cancer patients, predominantly within the first 1-3 weeks after surgery. These levels decreased to normal values within 4-6 weeks postoperatively. The median intraindividual coefficients of variation for the tumor markers ranged between 15% and 28% in 30 control women not having surgery. In general, it would seem advisable to wait 6 weeks after surgery before monitoring with CA 125 and TPA is started.


Protective effects of vitamin E against bleomycin-induced genotoxicity in head and neck cancer patients in vitro.

The quantitative protective effects of alpha-tocopherol acid and alpha-tocopherol acid succinate on mutagen-induced genotoxicity were studied in human lymphoblastoid cell lines and lymphocytes from cultures of peripheral blood. Both agents showed a dose-dependent protection both in lymphoblastoid cell lines and in lymphocytes of head and neck cancer patients. The concentrations used were in the phatmacologic concentration range of vitamin E. The protection against genetic toxic effects may be an important part of the cancer-protective effects of vitamin E.


[Neurectodermal malignant tumor of the soft tissues after treatment with immunosuppressive agents of lipoid nephrosis].

Immunosuppressive drugs are known to increase the risk of inducing neoplasia, especially acute leukaemia when high doses are used. A case of nephrosis in a 10 year-old boy treated with chlormethine (cumulative dose: 0.8 mg/kg) and chlorambucil (cumulative dose: 10 mg/kg) is reported. Four years after the beginning of the treatment an extraskeletal Ewing's sarcoma occurred. Since the review of literature failed to find any malignancy induced by such an immunosuppressive treatment for nephrosis, the question whether or not this extraskeletal Ewing's sarcoma was attributable to this treatment remains unanswered.


Induction of karyological damage by bleomycin in polyploidizating rabbit hepatocytes.

The effects of the antitumor drug bleomycin on polyploidizating hepatocytes of immature rabbits were investigated by combining cytochemical (static microfluorometry) and morphological (fluorescence and electron microscopy) approaches. In addition to a decelerating effect of the drug on the cell kinetics, there were several types of karyological damage (micronuclei, internuclear chromatin bridges, mitotic chromosome bridges), thus demonstrating a clastogenic activity of bleomycin on the DNA, Moreover, anomalous nuclear and cytoplasmic divisions suggest interference of the drug with other cellular structures (mainly the microtubule network) responsible for the cell kinetics.


Choroidal neovascularization associated with choroidal hemangiomas.

Two patients with choroidal hemangiomas developed choroidal neovascularization. One patient with Sturge-Weber syndrome, a unilateral diffuse choroidal hemangioma, and a serous detachment of the macula was treated with yellow dye laser photocoagulation in a grid pattern to the tumor before glaucoma filtration surgery. Four years after successful laser treatment, the patient developed subfoveal choroidal neovascularization in an area of treatment. A second patient with a circumscribed choroidal hemangioma developed spontaneous subfoveal choroidal neovascularization 12 years after initial diagnosis of the hemangioma. The development of choroidal neovascularization associated with choroidal hemangiomas represents a potential cause of poor visual outcome in these patients.