In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions. We have previously reported that cytokine- or LPS-activated human umbilical vein endothelial cell (HUVEC) monolayers secrete IL-8 that can act as a neutrophil-selective adhesion inhibitor. In our study we investigated the mechanisms involved in the leukocyte adhesion inhibitory action of IL-8. The leukocyte adhesion inhibitory effect appears to be mediated by the action of IL-8 on the neutrophil, does not involve down-regulation of relevant endothelial adhesion molecules such as endothelial-leukocyte adhesion molecule-1 or intercellular adhesion molecule-1, and is quantitatively similar in different endothelial activation states that are predominantly endothelial-leukocyte adhesion molecule-1 dependent or intercellular adhesion molecule-1 dependent. In addition to inhibiting the attachment of freshly isolated peripheral blood neutrophils to cytokine-activated HUVEC monolayers, IL-8 also promoted a rapid detachment of tightly adherent neutrophils from activated HUVEC, and abolished neutrophil transendothelial migration. Certain other chemoattractants, including FMLP and C5a, had similar inhibitory actions, indicating IL-8 was not unique in its ability to inhibit various neutrophil-endothelial interactions. In contrast, two other neutrophil agonists 1-0-alkyl-2-acetyl sn-glycero-3-phosphocholine and granulocyte-macrophage-CSF, which, like IL-8, are produced by activated HUVEC, as well as the leukocyte-derived chemoattractant leukotriene B4, exerted minimal inhibitory effects on adhesion. Regardless of their ability to modulate neutrophil-endothelial cell adhesion, all these agents induced altered leukocyte surface expression of functionally important adhesion molecules, including loss of L-selectin (leukocyte adhesion molecule-1, LECAM-1) and increase in CD11b/CD18. Thus, although the above agonists have been characterized primarily as chemoattractants, our findings demonstrate that these agents can exert a wide range of modulatory effects on neutrophil-endothelial adhesive interactions.


Analysis of HIV-induced autoantibodies to cryptic epitopes on human CD4. Antilymphocyte antibodies, including autoantibodies to CD4, have been reported in AIDS patients and are postulated to contribute to T cell depletion and immunologic dysfunction. In this paper, we characterize and localize binding sites of human anti-CD4 autoantibodies from a number of HIV+ patients. Epitope mapping by ELISA and Western blotting, together with cross-competition experiments, showed that common autoepitopes were localized to at least two topographically separate sites on the fourth domain of sCD4. These sites were partially dependent on the carboxyl terminus of the soluble molecule and were not exposed on full length membrane CD4, even under denaturing Western blotting conditions. Peptide screening identified peptides from the fourth and third domains that were recognized by several, but not all, anti-CD4 serum samples. Soluble CD4 affinity-purified antibodies were predominantly IgG1 and were not induced to bind mCD4 after gp120 binding to T cells. Analysis of HIV seroconversion panels showed that the appearance of anti-CD4 antibodies followed HIV seroconversion by 6 to 12 months and paralleled anti-gp120 reactivity. This suggested a correlation between immune reactivity to envelope and anti-CD4 antibody production. Together, the data indicate that human anti-CD4 antibodies recognize cryptic conformational and linear epitopes on a cleaved form of CD4. These findings suggest that HIV may induce abnormal cleavage of full length CD4, thereby exposing immunogenic self epitopes normally hidden from humoral and cellular immune interactions. This model of abnormal processing of self Ag has general implications for autoantigen exposure in other autoimmune disorders.


Human decidual leukocytes from early pregnancy contain high numbers of gamma delta+ cells and show selective down-regulation of alloreactivity. The mononuclear lymphoid cell population in human pregnant uterus mucosa, decidua, from early normal pregnancies was studied phenotypically and functionally. The phenotype was determined in situ by immunohistochemistry, and in isolated decidual mononuclear cell preparations by immunofluorescence and flow cytometry. A mild isolation procedure of gentle mechanical disruption followed by density gradient centrifugation was used. Leukocytes comprised a large part of the decidual tissue. They were present in aggregates mainly situated adjacent to the glandular epithelium. In addition, individual leukocytes were present intraepithelially, as well as scattered between the stromal cells and around vessels and lacunes. Four lymphocyte populations of approximately the same size were identified: TCR gamma delta+/CD56+ cells, TCR gamma delta+/CD56- cells, TCR gamma delta-/CD56+ cells, and TCR alpha beta+/CD8+ cells. TCR gamma delta- expressing cells comprised about 60% of the T cells. They were CD4-/CD8-, and about half of the TCR gamma delta+ cells expressed the memory/activation marker CD45RO. The Kp 43 Ag, earlier described on activated CD56+ and TCR gamma delta+ cells in peripheral blood, was essentially only expressed on the TCR gamma delta-/CD56+ cell population in decidua. At least 50% of the TCR alpha beta+ cells were CD8+. The function(s) of either one of these populations might be to prevent immunologic reactions against the fetus, to protect the uterus from unwanted extensive invasion of trophoblasts, or to protect the uteroplacental unit from infection. Decidual T cells did not respond to stimulation by alloantigens or mitogenic anti-CD3 mAb but responded to the same extent as PBMC to mitogenic lectins. The surface density of the TCR/CD3 complex was low on freshly isolated decidual lymphocytes, but could be up-regulated upon stimulation with PMA/Ionomycin. Local selective down-regulation of surface expression of the TCR/CD3 complex and of activation involving this complex might be one of the mechanisms by which a maternal immunologic reaction against the semiallogeneic fetus is prevented.


Tolerance mechanism in experimental ovarian and gastric autoimmune diseases. Neonatal splenocytes, neonatal thymocytes, or phenotypically mature adult thymocytes, transferred from normal BALB/c mice to syngeneic athymic nu/nu (or SCID) mice, led to autoimmune oophoritis and autoimmune gastritis, with corresponding serum autoantibodies, in the recipients. The overall disease incidence was 73%; the pathology ranged from mild to severe, with complete loss of ovarian follicles and gastric parietal cells. CD4+ neonatal spleen cells and CD4+ CD8- adult thymocytes were required for autoimmune disease induction. Adult spleen cells did not elicit disease, but they prevented disease when co-transferred with neonatal spleen cells. However, in confirmation of an earlier report by Sakaguchi et al., (J. Exp. Med. 161:72, 1985), a subset of adult splenic T cells expressing a low level of CD5 molecules elicited similar autoimmune diseases. Thus, self-reactive T cells responsible for autoimmune disease of the stomach and ovary are not effectively deleted in the thymus, and they exist in the peripheral lymphoid organs of normal mice. We conclude that the functional expression of the self-reactive T cells is ontogenetically regulated; whereas T cells in the neonatal mice readily elicited autoimmune diseases in nu/nu recipients, regulatory cells may render self-reactive T cells in the normal adults unresponsive.


Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen. Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.


Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions. The stability and recovery of six human recombinant cytokines (tumour necrosis factor (TNF), interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6) from whole blood was investigated with a view to optimizing blood collection and storage procedures prior to performing immunoassays. Blood from healthy volunteers was subjected to various processing and storage procedures. Blood samples were treated with either: ethylenediamine tetraacetic acid (EDTA) (1.5 mg/ml blood) (E); EDTA/Trasylol (1.5 mg and 1000 KIU/ml blood) (ET); heparin (30 IU/ml) (H) or allowed to clot (serum). The bloods were spiked with individual cytokines, split into aliquots and kept at 4 degrees C or RT. In the first instance spiked bloods from healthy volunteers (n = 5 per cytokine) were processed using sterile and non-pyrogenic materials and procedures. At regular time intervals, samples were cold spun, separated, flash frozen and assayed for the appropriate cytokine using RIA/IRMA methods. In a further study, timed separation was repeated with spiked blood from healthy volunteers (n = 5 per cytokine) using normal commercially available blood collection materials and procedures. In a third study, spiked blood from healthy volunteers (n = 3 per cytokine) was processed under sterile and non-pyrogenic conditions, and the blood samples separated, aliquoted and flash frozen within half hour of collection. These were then subjected to repeated cycles of freeze thawing at 4 degrees C or RT before assaying. In general, the stability of cytokines in whole blood was improved by storage at 4 degrees C and/or rapid separation. There was no significant difference between samples handled under sterile, non-pyrogenic conditions and those collected using normal blood collection procedures. The blood collection procedures described in this paper did not induce any of the six cytokines in the unspiked blood. Overall, EDTA-treated samples performed most consistently. The addition of trasylol did not significantly affect the results. Most of the cytokines appeared unaffected by up to three freeze thaw cycles. The stability and recovery of the spiked cytokines varied from least stable to most stable spiked cytokine as follows; TNF-alpha less than IL-6 less than IFN-gamma less than IL-1 alpha less than IFN-alpha less than IL-1 beta. The recovery of spiked IFN-gamma from heparinized plasma samples was considerably higher than any other plasma or serum samples. The recovery of spiked TNF-alpha and IL-6 from serum samples was consistently lower than amounts recovered from plasma samples (anticoagulant treated).(ABSTRACT TRUNCATED AT 400 WORDS)


A comparison of specific IgG antibody levels to the cell wall mannan of Candida albicans in normal individuals and in patients with primary antibody deficiency. An enzyme-linked immunosorbent assay (ELISA) has been developed to measure specific IgG antibody to the polysaccharide, cell wall mannan of Candida albicans (mannan). The results were expressed as arbitrary units/ml, with an inter- and intra-assay coefficient of variation of 7-11%. In establishing normal ranges we found that specific IgG to the mannan increased with age, with 18% of healthy children aged 3-10, 48% of healthy children aged 11-19 and 76% of an adult donor population having specific IgG antibody to mannan (greater than 30 U/ml). We have compared these normal ranges, with a group of patients with primary antibody deficiency (PAD). None of the 23 patients with PAD, which included common variable immunodeficiency, IgG subclass deficiency, and selective IgA deficiency, had titres greater than 30 U/ml. The patients with PAD had significantly lower levels of specific IgG anti-mannan antibody (median 9 U/ml) compared to healthy children aged 11-19 (median 26 U/ml) or adults (median 58 U/ml) (p = less than 0.001) but not children aged 3-10, (median 1 U/ml) (p = 0.08).


A monoclonal antibody-based enzyme immunoassay for human GMP-140/P-selectin. Two hybridoma cell lines producing monoclonal antibodies WGA-1 and PL7-6, reactive only with thrombin-stimulated human platelet have been established. Both these antibodies were investigated for their specific reactivity against GMP-140, based on the amino acid composition analysis of immunopurified antigen and N terminal amino acid sequencing of its protease fragments. A two-site enzyme immunoassay for quantification of human GMP-140 was developed using WGA-1 monoclonal antibody immobilized on 96-well microplates and horseradish peroxidase-labeled PL7-6 monoclonal antibody as detector. The assay was able to measure GMP-140 in serum and plasma with a sensitivity of about 5 ng/ml and a precision better than 10%. This assay will be useful for the detection of GMP-140 derived from platelets or endothelium in biological fluids and tissue extracts.


A monoclonal antibody recognising an epitope associated with pig interleukin-2 receptors. A monoclonal antibody is described which recognises an epitope associated with a receptor for interleukin-2 (IL-2) on pig lymphocytes. The monoclonal antibody inhibits high affinity binding of radiolabelled recombinant human IL-2 (rhIL-2) by pig lymphoblasts and also non-competitively inhibits both pig-TCGF and rhIL-2 maintained proliferation. By flow cytometry the antigen is apparently not present on freshly isolated blood lymphocytes but is detectable on small cells between 6 and 12 h after activation and on large cells by 24-h. These findings are comparable with those obtained using monoclonal antibodies recognising the 55 kDa alpha chain of the human and mouse IL-2 receptor (p55, TAC) expressed on activated cells in vivo and in vitro. However, the molecular weight of the porcine antigen is between 65 and 70 kDa.


P amylase is always greater than S in spot urine of normal subjects. Diagnostic implications. Single random samples of urine were collected from 50 control subjects; 27 patients with chronic pancreatitis; 19 with acute pancreatitis; 6 with acute on chronic pancreatitis; five in the recovery phase of acute attack; four patients with pseudocysts. Salivary (S) and pancreatic (P) amylase values were measured by cellulose acetate electrophoresis. The P amylase values always exceeded those of S amylase in the control specimens. In acute pancreatitis, both the lower and upper levels of total and P amylase were considerably higher than in the controls, and these high values tended to return to normal during the recovery phase of acute pancreatitis. The S amylase values were often very low or undetectable during the acute phase. Values for P amylase exceeded control values in patients with pseudocysts even in the presence of chronic pancreatitis. In chronic calcific pancreatitis, S amylase was higher than P amylase. We conclude that P amylase is always greater than S amylase in normal urine specimens, and a change in this pattern may be helpful in diagnosing various forms of pancreatitis.


Chromatographic characterization of insulin-like growth factor-binding proteins in human pregnancy serum. Insulin-like growth factor-binding proteins (IGFBPs) in maternal and umbilical cord sera have been analysed by combinations of gel filtration chromatography, affinity cross-linking and electrophoresis. On gel filtration chromatography, the majority of circulating IGF-I in non-pregnant and pregnant women was present in the large molecular mass (150 kDa) binding proteins (IGFBP-3). In umbilical cord serum, by contrast, most IGF-I was present in the 40 kDa binding proteins (consisting of IGFBP-1 and IGFBP-2). Western blots demonstrated an apparent progressive attenuation of IGFBP-3 and IGFBP-2 in serum from pregnant women with an increase in IGFBP-1. After prior cross-linking with disuccinimidyl suberate, the 150 kDa fractions (IGFBP-3) from non-pregnant and pregnant serum showed a similar pattern on SDS-PAGE (several bands at different molecular masses). However, IGFBP-2 (one of the components of the 40 kDa fractions) was undetectable, even after cross-linking, in serum from pregnant women later than 8 weeks of gestational age and in a mixture of maternal serum at term delivery and serum from non-pregnant women. This suggests that serum IGFBP-2 was degraded by specific proteases present in pregnancy serum. Following acid treatment, the 150 kDa fractions from pregnancy serum were split into smaller subunits or fragments while the 40 kDa fractions remained unchanged, suggesting that the 40 kDa binding proteins, are acid-stable. The present data demonstrate that IGFBP-3 is the principal IGFBP in pregnancy serum even though there is an apparent reduction in serum IGFBP-3 activity as revealed on Western blots. The absence of IGFBP-2 in serum from pregnant women may be due to degradation by proteases. In the fetal circulation IGFBP-1 and IGFBP-2 appear to be the major binding proteins for IGF-1.


Motor unit function during evolution of proximal axonal swellings. beta,beta'-Iminodipropionitrile (IDPN) impairs axonal transport of neurofilaments; their accumulation leads to the formation of proximal swellings in motor axons. Similar proximal swellings are a feature of some cases of motor neuron disease such as amyotrophic lateral sclerosis (ALS). Motor units in IDPN-treated animals were assessed to determine their relative susceptibilities to impaired function and whether the functional changes resulting from proximal axonal swellings share certain electromyographic features with ALS. Intrinsic properties of medial gastrocnemius motoneurones (MN) and contractile responses of their motor units were examined during the evolution of proximal axonal swellings in cats administered IDPN (50 mg/kg once weekly) for 7, 14 or 35 days. While conduction velocities were significantly decreased in all motor unit types by 35 days, the conduction slowing was greater in fast fatigable (types FF and FI) motor units than in fatigue resistant (types FR and S) motor units. Normal correlations between axonal conduction velocity and MN input resistance (Rin) and the inverse relationship between Rin and rheobase were lost with progression of the neuropathy. Twitch and maximum tetanic tension developed by fast-fatigable motor units declined early in the neuropathy, whereas fatigue-resistant units did not show similar changes until later stages of the intoxication. In some motor units, irregular and abnormal tetanic tensions were elicited by repetitive MN discharge. At 14 and 35 days, a novel, intermediate motor unit response classified as slow and fatigable (SF) was observed. Conduction block, characterized by repetitive MN firing without a corresponding contractile response, was observed in some type FF and S units by 35 days. Morphometric analysis of muscle fiber types showed significant atrophy, particularly in the type I fibers at 14-35 days; the atrophy reversed following cessation of IDPN administration. The influence of proximal axonal swellings on motor unit function in IDPN neuropathy is discussed in terms of reported electrophysiological alterations in motoneurone disease.


Effect of treatment on oligoclonal IgG bands and intrathecal IgG synthesis in sequential cerebrospinal fluid and serum from patients with subacute sclerosing panencephalitis. Oligoclonal IgG bands were analyzed in matching pairs of cerebrospinal fluid (CSF) and serum from 12 subacute sclerosing panencephalitis (SSPE) patients, using isoelectric focusing and immunofixation. Each patient was given isoprinosine, and four of the 12 patients were given alpha-interferon in addition. Two to 4 serial CSF and serum samples were collected from each SSPE patient during periods ranging from 1 to 16 months. In 3 SSPE patients a small number of new oligoclonal bands were seen in the follow-up CSF samples. In the other 9 SSPE patients there was no change in CSF band patterns between initial and follow-up specimens. Band patterns in serum remained unchanged between initial and follow-up samples. Although all 12 SSPE cases had higher IgG indices and increased rate of intra blood-brain barrier (BBB) IgG synthesis in comparison to patients with other neurological diseases, the values did not significantly differ between the first and follow-up specimens. We conclude that treatment of SSPE patients with isoprinosine or with isoprinosine and alpha-interferon had no significant effect on the CSF oligoclonal band profiles or IgG synthesis within the central nervous system.


Optic neuritis anti-myelin basic protein synthetic peptide specificity. Elevated titers of anti-myelin basic protein (anti-MBP) are highly associated with acute idiopathic unilateral optic neuritis as well as acute relapses of multiple sclerosis (MS). During acute phases of optic neuritis, free/bound antibody ratios are generally above unity, with high titers of free anti-MBP and relatively low or undetectable values of bound antibody. Three to 5 months after the acute phase when the majority of patients have recovered, free/bound anti-MBP ratios are below unity with low titers of free antibody and relatively higher levels of bound anti-MBP. Anti-MBP purified from cerebrospinal fluid of patients with optic neuritis are neutralized by synthetic peptides of human MBP containing overall amino acid residues 61-106 and do not react with synthetic peptides corresponding to residues 1-60 and 107-170. Anti-MBP may either have multiple epitopes in the region corresponding to residues 61-106 or it may react with a discontinuous epitope in this range. The mechanism of the optic nerve demyelination may be associated with anti-MBP binding in situ to MBP in the 61-106 amino acid region.


Vascular smooth muscle hyperplasia underlies the formation of glomeruloid vascular structures of glioblastoma multiforme. The origin of the vascular hyperplasia seen in glioblastoma multiforme is a matter of debate. To test the predominant hypothesis that these glomeruloid structures are of endothelial origin the following study was undertaken. Seven glioblastomas containing prominent glomeruloid vascular structures were stained with Ulex europaeus agglutinin I (UEA-1) and with antibodies against factor VIII/related antigen (fVIII/RAg), glial fibrillary acidic protein (GFAP), S-100 protein, muscle specific alpha-actin (MSA) and smooth muscle specific alpha-actin (SMSA). The GFAP and S-100 antibodies stained the neoplastic glial component of each tumor but did not bind to vascular cells. Endothelial cells lining the lumina of normal vessels and the lumina of glomeruloid vascular structures stained positively with both UEA-1 and fVIII/RAg antibody. No other cells were found to be stained by UEA-1 or by fVIII/RAg antibody. Smooth muscle cells of the normal vasculature stained positively exclusively with anti-MSA and anti-SMSA antibodies. The same pattern of positive actin antibody staining was seen in the majority of cells forming the glomeruloid structures; however, the cells lining the vascular lumina did not bind the MSA and SMSA antibodies. These data strongly suggest that the vascular proliferation resulting in glomeruloid structures is due in large measure to smooth muscle hyperplasia.


Smooth muscle can comprise the sarcomatous component of gliosarcomas. The sarcomatous component of gliosarcomas is thought by many to originate from the vascular proliferation seen in glioblastoma multiforme and has, therefore, been assumed to be endothelial. Immunohistochemical staining of four gliosarcomas has led us to an alternate theory. Pathologically all four tumors were composed of at least two cell types; the first had a stellate, glial appearance and the second was either spindled or polygonal in shape. Polygonal cells were associated with glomeruloid vascular structures in some areas. Both components of each neoplasm were cytologically malignant. Glial fibrillary acidic protein and S-100 antibodies stained most of the glial-appearing cells and some of the spindled cells, but not the polygonal cells. Muscle specific alpha-actin and smooth muscle specific alpha-actin antibodies stained only the malignant spindled and polygonal cells and normal vascular smooth muscle. Ulex europaeus agglutinin I and anti-factor VIII/related antigen antibody stained only cells lining vascular lumina. The staining results suggest that the malignant mesenchymal component of these tumors is of smooth muscle origin. Having demonstrated elsewhere that glomeruloid vascular structures of glioblastoma multiforme contain smooth muscle cells, we propose here that gliosarcomas can represent one end of the spectrum of glioma-induced vascular smooth muscle proliferation.


Progressive hind limb paralysis in mice carrying a v-Mos transgene. To study the function of the protooncogene Mos in mouse brain development we have created a transgenic mouse model system in which an activated form of the gene, the murine retroviral v-Mos gene, is highly overexpressed in the brain. Six transgenic founder animals and mice of one established transgenic line (line TG66) displayed a progressive hind limb paralysis with onset between 18 days and 9 months. The severity of the neurological phenotype correlated with pathological alterations and the degree of v-Mos expression in the brain which varied between individual animals of line TG66. The most striking feature of the brain pathology was the presence of large, abnormal astrocytes in the cerebellum, medulla, thalamus and in the dorsal horn of the spinal cord. These areas also contained shrunken and basophilic neurons whose cytoplasm was abnormally immunoreactive for phosphorylated epitopes of neurofilaments. In addition to neuropathologic changes, these mice also displayed aberrant eye lens differentiation and absence of hair cells in the inner ear. These results establish v-Mos transgenic mice as a model system to study progressive neurodegenerative disease and provide further evidence that the Mos protein-serine/threonine kinase has a function in brain development.


Peripherin and neurofilament protein coexist in spinal spheroids of motor neuron disease. We have compared the immunolocalization of neurofilament protein (NF) with two other neuronal-specific intermediate filament proteins in large spinal axonal swellings (spheroids) of motor neuron disease and controls. All spheroids labeled each of the three different subunits of NF, the low, middle, and high molecular weight polypeptides. In doublelabel immunofluorescence, 300 axonal swellings were immunostained for NF, and 87% of them contained the intermediate protein peripherin. The pattern of immunostaining of NF and peripherin was indistinguishable at a high resolution viewed in 1 microns optical sections by confocal microscopy. A minority of the spheroids contained alpha-internexin, another intermediate protein, but it was weakly immunoreactive. The immunostaining of axonal swellings was similar for all epitopes tested in motor neuron disease and control subjects. The findings suggest that peripherin as well as neurofilament protein are major components of the proliferated intermediate filaments in spheroids.


Expression of mRNA for glial fibrillary acidic protein after experimental cerebral injury. This study was undertaken to determine whether a mRNA for glial fibrillary acidic protein (GFAP) was present in increased amounts as a response to injury and, if so, how was its temporal expression related to the demonstration of GFAP by immunocytochemical techniques. A cerebral freeze-injury was produced in mice and at intervals thereafter the animals were anesthetized, perfused with formalin and histological sections of the brain through the injured area were prepared. A riboprobe for GFAP mRNA labeled with S35 and an immunocytochemical probe for GFAP were utilized to localize mRNA and GFAP immunoreactivity, respectively. For mRNA studies, the histological slide exposed to either sense or antisense probe was overlaid with x-ray film or dipped in photographic emulsion. The developed film was quantitated by digital image analysis. Emulsions were examined by dark-field microscopy. The results indicate that mRNA for GFAP is increased in the cortex in the environs of the injury by 6 hours, becomes maximal at 4-5 days, and is present in increased amounts up to 14 days. The message is enhanced in the adjacent cortex, the subpial region, the adjacent corpus callosum and in the ipsilateral and contralateral callosal radiations. This pattern of enhancement follows the distribution of post-injury edema. Glial fibrillary acidic protein is demonstrable at 24-48 hours after injury. Thus, there is a rapid response of the astrocyte to injury with increased mRNA expression that is followed by expression of GFAP immunoreactivity.


Shunting of excitatory input to dentate gyrus granule cells by a depolarizing GABAA receptor-mediated postsynaptic conductance. 1. Stimulation of the perforant path in the outer molecular layer of the adult rat dentate gyrus produced a depolarizing post-synaptic potential (DPSP) in granule cells when recorded using whole-cell techniques in the standard hippocampal slice preparation at 34 degrees C. The postsynaptic currents (PSCs) contributing to the DPSP were analyzed using specific receptor antagonists in current- and voltage-clamp recordings. 2. The DPSP reversal potential was dependent on the intracellular chloride concentration, and the amplitude of the DPSP was increased 55% after perfusion of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI). The GABAA receptor-mediated PSC reversed at -66 mV, which was 19 mV positive to the resting membrane potential (-85 mV) but hyperpolarized relative to action potential threshold. At -35 mV, the GABAA PSC had a latency to peak of 12.9 ms after the stimulus and decayed monoexponentially with an average time constant of 23.4 ms. 3. The component of the PSC blocked by the Quis/AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) had a latency to peak of 7.1 ms and decayed monoexponentially with a time constant of 9.9 ms at -35 mV. The N-methyl-D-aspartate (NMDA) receptor-mediated PSC, which was blocked by D-amino-5-phosphonovaleric acid (D-AP5), had a waveform that was similar to the GABAA PSC: the latency to peak was 16 ms and the decay was monoexponential with a time constant of 24.5 ms at -35 mV. 4. The ratio of the peak PSCs mediated by GABAA, Quis/AMPA, and NMDA receptors measured at -35 mV with cesium gluconate electrode solutions was 1:0.2:0.1. This ratio was essentially constant over the range of stimulus intensities that produced compound PSC amplitudes of 80-400 pA. 5. Measured at its reversal potential, the GABAA receptor-mediated postsynaptic conductance (GGABA-A) decreased the peak DPSP amplitude by 35%, shunted 50% of the charge transferred to the soma by the excitatory PSC, and completely inhibited the NMDA receptor-mediated component of the DPSP. 6. Simultaneous stimulation of presynaptic fibers from both the perforant path and interneurons results in a large depolarizing GGABA-A that inhibits the granule cell by shunting the excitatory PSCs. As predicted by models of shunting, the similar kinetics of the GABAA and NMDA PSCs leads to particularly effective inhibition of the NMDA PSC. The more rapid Quis/AMPA PSC is less affected by the GGABA-A, so that granule cell excitation under these conditions is primarily due to Quis/AMPA receptor activation.


Modulation of excitatory amino acid responses in rat dorsal horn neurons by tachykinins. 1. In freshly isolated spinal dorsal horn (DH) neurons (laminae I-III) of the young rat, the effects of tachykinins (substance P, neurokinin A) on inward current induced by excitatory amino acids were studied under whole-cell voltage-clamp conditions. 2. When the cells were clamped to a holding potential of -60 mV, a simultaneous application of N-methyl-D-aspartate (NMDA) (10(-4) M) and substance P (SP) (2 x 10(-9)-10(-7) M) for 10 s reversibly enhanced (by 129.6 +/- 8.2%, mean +/- SE) the peak amplitude of the initial transient component of the NMDA-induced current in approximately 60% of the examined cells and reduced it (to 83.3 +/- 2.7%) in 27% of the cells. In addition, SP produced an increase (by 133.6 +/- 11.7%) or a small decrease (to 85.9 +/- 1.4%) in the steady-state component of the NMDA response. In difference to SP, a simultaneous application of NMDA (10(-4) M) and neurokinin A (NKA) (10(-10)-10(-7) M) reversibly suppressed (to 86.8 +/- 2.1%) the peak amplitude of the NMDA-induced current in 75% of the examined cells. 3. The NMDA-induced currents were modulated by tachykinins not only during the coadministration but up to 20 min after the removal of the peptide. SP potentiated the initial peak NMDA current by 147.9 +/- 8.1% in 78% of examined cells and decreased it (76.3 +/- 5.7%) in 11% of cells. The potentiating effect was concentration-dependent (range: 10(-11)-10(-8) M) and reversible, but it was reduced with repeated applications. In addition, SP increased (by 125.4 +/- 3.6%) or reduced (to 86.0 +/- 1.8%) the steady-state component of the NMDA response. 4. When the single DH neurons were exposed to SP or NKA for 30 s-7 min before the testing of the NMDA responses, tachykinins had two distinct effects on the peak amplitude of the transient component of the NMDA-induced current, consisting of an initial depression (SP: to 64.8 +/- 2.1%; NKA: to 76.3 +/- 4.4%) followed by a potentiation (SP: by 146.6 +/- 6.8%; NKA: by 178.4 +/- 35.2%). The enhancing effect in some cells lasted less than or equal to 1 h. 5. A claimed novel nonselective tachykinin antagonist, spantide II (10(-8) M) coadministered with NMDA (10(-4) M), slightly depressed the peak component of NMDA-induced current. In addition, it effectively blocked the SP-induced potentiation of the responses of DH neurons to NMDA.(ABSTRACT TRUNCATED AT 400 WORDS)


Charybdotoxin and apamin sensitivity of the calcium-dependent repolarization and the afterhyperpolarization in neostriatal neurons. 1. Intracellular recordings from neostriatal neurons in an in vitro slice preparation of the rat brain were used to analyze the pharmacological sensitivity of the action potential (AP) repolarization and the afterhyperpolarization (AHP) that follows a single action potential. The interspike voltage trajectory and the AHP could be divided into two main parts: a fast component lasting a few milliseconds and better observed during a train of spikes, and a slow component lasting approximately 250 ms and that comprises the main portion of the AHP. In some cells, a slow (up to 1 s) component of low amplitude was also detected. 2. Single APs were elicited at two imposed membrane potentials (around -60 and around -80 mV). The AP amplitude was larger, the repolarization rate was faster, and the duration was shorter when spikes were evoked at -80 mV. When measured from the -60 mV holding potential, the afterpotential was an AHP with peak amplitude of -5 mV. The afterpotential became a delayed depolarization (DD) at -80 mV. 3. Firing frequency adaptation was voltage sensitive. The firing of APs induced by long intracellular current pulses from a holding potential of -80 mV exhibited only a slow-frequency adaptation (time constant of seconds). However, at -60 mV, an initial and faster frequency adaptation was evident (time constant of tens of milliseconds). 4. The Ca2+ channel blocker Cd2+ retarded AP repolarization rate. This effect correlated with a significant block of the fast and slow components of the AHP. In contrast, Ni2+ had no significant effects on the same parameters.(ABSTRACT TRUNCATED AT 250 WORDS)


NMDA receptor-mediated currents are prominent in the thalamocortical synaptic response before maturation of inhibition. 1. The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) is thought to underlie synaptic plasticity in both adult and developing CNS; however, its involvement in the thalamocortical synapse has not yet been directly demonstrated. 2. Whole-cell, thalamus-evoked synaptic currents were recorded from layer IV cells in slices of immature mouse somatosensory cortex. 3. Earlier than postnatal day 9 the majority of responses were monosynaptic and purely excitatory, with both non-NMDAR and NMDAR-mediated glutamatergic components. 4. In older animals, disynaptic inhibitory currents summated with the excitatory ones and lowered the reversal potential of the response to voltages at which the NMDAR conductance is mostly blocked. 5. These findings suggest a cellular basis for the transient plasticity observed in layer IV during early postnatal development.


Histamine activates chloride conductance in motor neurons of the lobster cardiac ganglion. 1. Individual motor neurons of the lobster cardiac ganglion were voltage clamped with two microelectrodes. Superfusion of histamine evoked a concentration-dependent membrane current. The mean effective concentration (EC50) for the concentration-effect relationship was 28 microM. 2. The amplitude and polarity of the histamine-activated current depended on intracellular and extracellular Cl- concentration. The membrane potential at which the current polarity reversed was a function of the Cl- equilibrium potential. 3. The histamine-activated Cl- conductance was voltage dependent, increasing with depolarization. As a consequence, the histamine-evoked current showed outward rectification. 4. We conclude that histamine activates a Cl- conductance with biophysical properties similar to the crustacean Cl- conductance activated by gamma-aminobutyric acid (GABA) and to the histamine responses described in lobster olfactory and stomatogastric neurons. 5. The response to histamine was competitively inhibited (IC50 = 7 microM) by cimetidine, an H2 subtype inhibitor in mammals. Ranitidine, pyrilamine, chlorpheniramine, diphenhydramine, and cyproheptadine were 50-100 times less potent than cimetidine. Tubocurarine, a Cl- channel blocker, blocked with an IC50 of 20 microM, but picrotoxin did not begin to inhibit the histamine response until concentrations exceeded 0.1 mM. 6. These results suggest that the response cannot easily be classified with the use of the pharmacological categories developed in mammals. Like the Cl(-)-dependent responses to various neurotransmitters in a number of invertebrates, the histamine response in the lobster cardiac ganglion was inhibited by tubocurarine. 7. Both GABA and histamine had similar effects on the motor neurons, but only GABA inhibited pacemaker bursts. In this respect, GABA more resembles the endogenous inhibitory postsynaptic potential.(ABSTRACT TRUNCATED AT 250 WORDS)


Cellular localisation of c-myc product in human colorectal epithelial neoplasia. Aberrant expression of c-myc has been implicated in the development of colorectal carcinomas. We have used monoclonal antibodies 6E10 and 9E10, raised against mid-sequence and C-terminal peptides of the c-myc protein, to study the distribution of myc protein in normal and diseased bowel at the light microscope and ultrastructural levels. Normal mucosa showed staining only of some nuclei in the proliferative zones of crypts. In adenomas, staining varied from predominantly nuclear to pancellular to focal or pancytoplasmic. Moderately well differentiated areas of carcinomas gave strong focal cytoplasmic staining, while in poorly differentiated tumours staining was pancytoplasmic. Electron microscopy with these antibodies detected myc protein associated with dense chromatin and, where cytoplasmic staining occurred, with polyribosomes. Tumours showed a reduced staining of nuclear pores compared with normal tissue. Comparison of staining patterns with 6E10 and 9E10 in normal tissue, adenomas, and tumours suggests that tumour progression is associated with an accumulation of cytoplasmic c-myc protein, perhaps resulting from alterations to the C-terminus which reduce the efficiency of nuclear targeting of the protein and thus disrupt the regulation of the cell cycle.


P53 protein expression in lymphomas and reactive lymphoid tissue. P53 is a tumour suppressor gene, located in the short arm of chromosome 17, which encodes for a nuclear protein involved in the control of cellular growth, regulating the entry of the cell into the S-phase. P53 mutations have been identified in a progressively increasing number of human malignancies. Nuclear p53 protein is usually present in non-tumour cells in minute concentrations, due to its short half-life. In contrast, tumours with p53 mRNA mutations show a higher nuclear protein concentration, detectable by immunohistological techniques, due to stabilization by complexing with other proteins such as heat-shock protein or wild-type p53 protein. Levels of nuclear p53 protein detected by immunohistochemistry with the monoclonal antibody PAb 1801 were measured with the aid of an image analysis system in 83 non-Hodgkin's lymphomas (NHLs) and 13 cases of Hodgkin's disease, as well as in 14 cases of normal thymus, reactive tonsils, and lymphadenitis. High levels of p53 protein (greater than 5 per cent of the cells) were present only in high-grade lymphomas (in the proportion 13/55), with a peak incidence in Burkitt's lymphoma (5/8 cases). Lower levels (less than 5 per cent) of p53 protein were detected in low-grade B- and T-cell lymphomas, as well as in most of the cases of Hodgkin's disease, where p53 protein was selectively present in Hodgkin and Reed-Sternberg cells. In 5/14 reactive tonsils or lymph nodes, occasional p53-positive cells were identified. These results suggest a relationship between levels of p53 protein and the aggressiveness of NHL.(ABSTRACT TRUNCATED AT 250 WORDS)


Lymphocytes in pseudomembranes of late prosthetic joint failure. The pseudomembrane formed in association with late aseptic prosthesis failure contains a mixed giant cell and histiocytic infiltrate with variable numbers of lymphocytes. Immunolabelling with a panel of antibodies on paraffin sections was undertaken to define the nature of the lymphoid infiltrate in 19 cases. In all cases, the predominant lymphoid cell was a memory (CD45RO+, CD45RA-) T-cell. B-cells were rare. Tissue from patients with rheumatoid arthritis (RA) contained greater numbers of T-cells when compared with patients with osteoarthritis (OA), suggesting that the intensity of the lymphoid infiltrates reflects the underlying joint disease rather than necessarily being part of a hypersensitivity response to wear debris.


An immunohistological study of cartilage and synovium in primary synovial chondromatosis. The antigenic phenotype of cartilage and synovial cells from six cases of primary synovial chondromatosis (PSC) was determined. This was compared with profiles similarly obtained for adult and fetal cartilage cells. The Ki-67 (proliferation-associated) antigen was present on 40-50 per cent of chondrocytes in the proliferative zone of fetal epiphyseal cartilage but absent in chondrocytes of adult articular cartilage and PSC cartilage nodules. The absence of Ki-67 antigen suggests that there were no proliferating cells in the synovium or cartilage in these cases of PSC. In PSC alone, some chondrocytes in the cartilage nodules and mononuclear subintimal cells around the nodules also reacted for CD68, suggesting that growth of the cartilage nodules may occur by a metaplastic process. All synoviocytes in PSC were positive for leucocyte common antigen, HLA-DR, and CD68, a pattern typical of reactive rather than normal synovium.


Tissue-type plasminogen activator and its inhibitor in human glomerulonephritis. We carried out an immunohistochemical study of tissue-type plasminogen activator (PA) and urokinase-type PA, and their inhibitors, PA inhibitor-1 and PA inhibitor-2, using renal biopsy specimens obtained from 86 patients with various forms of glomerulonephritis. The controls were four normal renal tissue specimens. On immunofluorescent observation, granular staining for tissue-type PA was found to be distributed along the glomerular capillary walls. The fluorescence was weak in the normal renal tissue and occasionally intense in the tissues of patients with IgA nephritis, minimal change nephrotic syndrome, and lupus nephritis. PA inhibitor-1 was abundant in the glomerular epithelial cells and scarce in the mesangial area and glomerular capillary lumens of the normal renal tissues. This was confirmed by immunoelectron microscopy using gold staining. The fluorescence of PA inhibitor-1 was weaker in some specimens of nephritic tissues than in the normal renal tissues. Urokinase-type PA and PA inhibitor-2 were negative within the glomeruli in all the specimens. In the glomerulonephritic tissues which were fibrin deposition-positive, tissue-type PA expression in the glomeruli tended to be strong. An association between fibrin deposition and PA inhibitor-1 staining was not clear. These data suggest that expression of tissue-type PA in the glomeruli increases in association with fibrin deposition.


Immunolocalization of regenerating cells after submassive liver necrosis using PCNA staining. Little data exist on the proliferative state of liver cells and its relationship with various morphological findings in acute liver failure (ALF) in man. In this study we used the monoclonal antibody NCL-PCNA (clone PC-10) against the proliferating cell nuclear antigen (PCNA) to detect cycling cells in paraffin sections of 3 normal livers, 14 post-mortem needle-specimens of submassive hepatic necrosis (SHN) due to paracetamol overdosage (POD), and 10 hepatectomy specimens obtained at transplantation in patients with acute or subacute liver failure of presumed viral aetiology. In normal livers, only occasional sinusoid-lining cells were stained, whereas in SHN following POD or presumed viral hepatitis, hepatocytes of variable morphology showed significant immunoreactivity. Following POD, immunoreactivity was higher in samples taken within 5-6 days than in those obtained at 9-11 days, a pattern reminiscent of the decrease in the rate of regeneration, previously documented after partial hepatectomy in humans. Immunolabelled hepatocytes were aggregated in multiacinar 'nodules' in cases with a map-like distribution of collapsed and non-collapsed parenchyma. Ductules demonstrated comparatively less staining, but extensive labelling was exceptionally found in areas of complete hepatocellular dropout. In these areas, small elongated cells with strongly PCNA-positive ovoid nuclei, forming periportal sprouting cords or incorporated into the lining of ductules, were most remarkable in that they closely resembled 'oval cells' described in animal studies.


The distribution of LH39 basement membrane epitope in the tumour stroma of oral squamous cell carcinomas. LH39 monoclonal antibody detects a novel component of epithelial and endothelial basement membranes. The expression of LH39 antigen was investigated by immunohistochemistry in 55 oral squamous cell carcinomas and compared with 15 pyogenic granulomas of skin and oral mucosa, 20 non-specific oral ulcers, and 20 specimens of normal oral mucosa. The distribution of this basement membrane epitope was compared with that of other basement membrane components, type IV collagen, and laminin. LH39 monoclonal antibody labelled basement membrane surrounding small blood vessels in normal human organs. In oral squamous cell carcinomas, in contrast to the other basement membrane antigens, the LH39 epitope was not detectable in vessels within histologically recognizable tumour stroma. Neovascularization is known to attend malignant neoplasms and this finding was interpreted as absence of LH39 antigen within newly formed vessels. In support of this hypothesis, LH39 immunoreactivity was absent in newly formed blood vessels within pyogenic granulomas and the granulation tissue within ulcers. As increasing neovascularization is reported to correlate with a rising rate of metastasis, assessment of tumour angiogenesis may be of value in selecting patients for initial aggressive therapy.


Assessment of p53 protein expression in normal, benign, and malignant oral mucosa. Recent studies have shown the accumulation of high levels of p53 protein to be associated with malignant disease, within a range of tissues. This paper assesses p53 expression in oral mucosal disease. Biopsies were obtained from a range of oral disorders which included normal, benign, premalignant, and malignant oral tissue. In addition, oral smears were obtained from a limited number of patients with biopsy-proven oral cancer. Expression of the p53 protein was assessed using the polyclonal antibody CM1, together with a standard immunoperoxidase technique. A total of 37 oral cancers were assessed, of which 20 were found to express the p53 protein (54 per cent of cases). The p53 protein was not identified in normal, benign, or premalignant oral mucosa (54 cases). The identification of p53 within biopsies of oral mucosal lesions would appear to correlate with oral malignancy.


Epstein-Barr virus latent membrane protein expression by Hodgkin and Reed-Sternberg-like cells in acute infectious mononucleosis. In the light of reports of latent membrane protein (LMP) expression by Hodgkin and Reed-Sternberg (HRS) cells, paraffin sections of tonsil (two cases), lymph nodes (eight cases; three cervical, one axillary, and four inguinal) and spleen (four cases) from 14 patients with acute infectious mononucleosis (IM) have been examined for the presence of HRS-like cells and immunostained with an antibody to LMP. Sections of the tonsils and one lymph node were also stained with a panel of antibodies which characterize HRS cells of Hodgkin's disease. The tonsils contained abundant HRS-like cells, mainly adjacent to the crypts, which were highlighted by strong LMP expression. The immunophenotype of these cells closely, but not completely, resembled that of HRS cells of Hodgkin's disease. The lymph nodes and spleens showed the typical changes of acute IM but only few LMP-positive HRS-like cells were present in the cervical lymph nodes and hardly any were present in the inguinal nodes and spleen. These findings suggest that tonsillar crypt squamous epithelium may play a role in the formation of LMP-positive HRS-like cells; these cells could be progenitors of Hodgkin's disease HRS cells and, if so, this might explain the restricted sites of presentation of Hodgkin's disease.


Mitochondrial and chromosomal DNA alterations in human chromophobe renal cell carcinomas. Renal cell tumours are characterized by the loss of chromosome 3p and trisomy of 5q segments (common, non-papillary renal cell carcinoma), or by trisomy of chromosomes 7 and 17 and loss of the Y chromosome (papillary renal cell carcinoma), or by random karyotype changes and mitochondrial DNA alterations (renal oncocytoma). We have studied by means of RFLP analysis the genomic and mitochondrial DNA in 11 chromophobe renal cell carcinomas, which have a unique morphology among kidney cancers. We found a loss of the constitutional heterozygosity at chromosomal regions 3p, 5q, 17p, and 17q, a combination of allelic losses that has not been found in other types of renal cell tumours. Three of the tumours showed a gross alteration in the restriction pattern of the mitochondrial DNA. A combined morphological and genetic analysis suggests that chromophobe renal cell carcinoma is a distinct entity.


Parathyroid glands in primary hyperparathyroidism: an ultrastructural morphometric study of 25 cases. In parathyroid glands removed from patients with primary hyperparathyroidism (pHPT), hyperplasias and adenomas cannot be distinguished from one another by light microscopy when only one gland is available for examination. When a second gland is available, it is necessary to establish whether it is normal, suppressed, or hyperplastic. This distinction may be difficult, and the main criterion is the amount of cytoplasmic lipid in the parenchymal cells. If the lipid is abundant, the gland is considered normal or suppressed, and if it is scanty, the gland is interpreted as hyperplastic. We have performed a morphometric ultrastructural study to test the reliability of this criterion. Twenty-five adenomatous glands removed from patients with pHPT, when compared with glands of normal size from euparathyroid patients, showed a significant increase in the parameters indicative of metabolic activity, namely the size of the Golgi apparatus, the amount of rough endoplasmic reticulum, and the length of plasmalemmas. In addition, the amount of cytoplasmic lipid was significantly reduced. Furthermore, 25 glands of normal size removed from the same patients with pHPT showed an amount of lipid similar to that of normal glands from euparathyroid patients. However, all the parameters indicative of metabolic activity were significantly higher than those in glands from euparathyroid patients and comparable to those found in adenomatous glands. These results suggest that in pHPT, normal-size glands are as active as adenomatous glands, regardless of a higher lipid content.


Flow cytometry of oesophageal mucosal biopsies; epidermal growth factor receptor, and CD15. Flow cytometry may be used to examine the properties of single or isolated cells. We have shown that it is possible to disaggregate and label oesophageal epithelial cells for two surface markers, CD15 and epidermal growth factor receptor. We have previously demonstrated these markers in oesophageal squamous cells using immunoperoxidase techniques. These labelled disaggregated cells could then be measured by flow cytometry.


Differential expression of integrin alpha chains by renal epithelial cells. We have investigated the expression of integrin chains by human renal epithelial cells during in vivo renal differentiation and in vitro cell culture on different extracellular matrices. Using the immunoperoxidase technique to visualize the binding of monoclonal antibodies to different integrin chains in fetal and adult kidneys, we found a change during development from alpha 1 beta 1, alpha 3 beta 1, and alpha 4 beta 1-positive blastemal cells to alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1-positive epithelial cells. The pattern of integrin expression correlates with the presence in the extracellular matrix of the appropriate ligands. In in vitro cell culture experiments, renal epithelium expressed alpha 3 and alpha 5 integrins on all extracellular matrices. Integrins alpha 2 and alpha 6 were found only in cells grown on a laminin-containing substratum. Fibronectin and alpha 5 integrin co-localized on the ventral surface of cells grown on a laminin substratum and at the periphery of cells on glass coverslips. These results suggest that there is a close relationship between integrin alpha chain usage and the presence of appropriate ligands in the extracellular matrix.


Collagen composition and ultrastructure of the so-called amianthoid fibres in palisaded myofibroblastoma. Ultrastructural and immunohistochemical study. A very prominent feature of the recently described intranodal palisaded myofibroblastoma is the occurrence of stellate crystalline extracellular deposits known as 'amianthoid fibres', and thought to represent abnormally thick collagen fibrils. We showed at the ultrastructural level that these deposits are composed of fibrils with the periodicity and width typical of normal native collagen. Using antibodies directed against various extracellular components, we have determined that these structures contain type I and III collagens. Our results indicate that the crystalloid deposits in intranodal palisaded myofibroblastoma called 'amianthoid fibres' do not contain giant collagen fibrils widely regarded as characteristic of the morphological concept 'amianthoid' in other locations.


Characterization of HIV isolates arising after prolonged zidovudine therapy. Human immunodeficiency virus type 1 (HIV-1) was isolated from five patients with late-stage disease treated with zidovudine (ZDV) for more than 1 year. Peripheral blood mononuclear cells (PBMCs) were used for all virus isolations and to assay for drug resistance. The isolates exhibited a 10- to 100-fold decrease in ZDV susceptibility compared to pretreatment isolates. Multiple clones of a 618 bp segment of the HIV reverse transcriptase gene encompassing codons 60-250 were sequenced for each isolate. The association of alterations at codons Asp67----Asn, Lys70----Arg, Thr215----Phe or Tyr, and Lys219----Gln with ZDV resistance has been previously noted (ref. 5). In this study, the most frequent alterations was Thr215----Tyr although genotypic mixtures of Thr/Tyr and Phe/Tyr were also observed. One isolate with a Tyr215 alteration and unaltered codons at 67, 70, and 219 had high-level ZDV resistance. Alterations at codons 67, 70, and 219 did not appear to increase resistance when seen in combination with Tyr215. Virus isolates obtained from each patient by cultivation with either 0 or 4 microM ZDV were compared and found to have similar alterations at codons 67, 70, 215, and 219, although one instance of apparent in vitro selection for Tyr215 over Phe215 was observed. Assays using PBMCs for virus propagation will permit susceptibility testing of HIV isolates from most patients on antiretroviral drugs to investigate the clinical significance of drug resistance.


Concentrations of major plasma proteins in serum and whole-tissue extracts of porcine fetuses during development. In extracts from fetuses up to 32 days of gestation, the major serum proteins were fetuin, alpha-fetoprotein and alpha 1-antitrypsin, but albumin was not detected. The concentration of all proteins rose with age until 40-50 days of gestation; and then the serum concentration of alpha-fetoprotein (2.9 mg ml-1), alpha 1-antitrypsin (4.4 mg ml-1) and transferrin (2.6 mg ml-1) fell progressively to about 1 mg ml-1 at birth, whereas those of fetuin, albumin and alpha 1-acid glycoprotein increased. The patterns of serum proteins in fetuses at about the middle of gestation were similar in extracts and sera. At birth, the major proteins were alpha 1-acid glycoprotein and fetuin, which accounted for 45 and 18% of serum proteins, respectively. Albumin represented only 7% of serum proteins at this age. For most of the second gestational period, the six quantified proteins accounted for about 85% of total serum proteins. In early gestation, a significant proportion of serum proteins was intracellular.


Viable embryos and normal calves after nuclear transfer into Hoechst stained enucleated demi-oocytes of cows. Bovine oocytes were bisected, stained with Hoechst 33342 and observed under a fluorescent microscope to identify nucleated and enucleated demi-oocytes. Other oocytes were bisected but not stained, or bisected and only half of each oocyte stained, and viewed under a fluorescent microscope. The oocytes were then used for nuclear transfer by fusing them with embryonic blastomeres from a 5-6 day bovine embryo. The fusion rate and proportion developing into compact morulae or blastocysts was compared among different types of demi-oocytes. Expt 1 examined the effect of staining and indicated no effect on either fusion rate or embryonic development whether or not the oocytes were stained. In Expt 2, stained and unstained nucleated and enucleated oocytes were compared. As in the first experiment, there were no differences between stained and unstained demi-oocytes. There was no difference between fusion rates of nucleated and enucleated oocytes. However, there was a significant difference in embryonic development between nucleated (10.4%) and enucleated (22.6%) demi-oocytes (P less than 0.05). In a final experiment, stained and unstained enucleated oocytes were used for nuclear transfer and the resulting embryos transferred into recipient cows. There was no difference in pregnancy rates or in the number of normal calves born whether stained or unstained recipient oocytes were used. Results from these experiments indicate that Hoechst staining and fluorescent microscopy can be used to identify enucleated demi-oocytes, and that these can be used for nuclear transfer, and result in viable embryos and normal calves.(ABSTRACT TRUNCATED AT 250 WORDS)


Motility characteristics and membrane integrity of cryopreserved human spermatozoa. The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.


Nine cystic fibrosis patients homozygous for the CFTR nonsense mutation R1162X have mild or moderate lung disease. The clinical course of nine cystic fibrosis patients homozygous for the CF gene nonsense mutation R1162X was investigated. Since this mutation should lead to an interruption in the synthesis of the cystic fibrosis transmembrane regulator (CFTR) protein, a severe clinical course was expected. All patients showed pancreatic insufficiency, while the course of the lung disease was mild to moderate. These results suggest that this form of truncated CFTR protein, still containing the regulatory region, the first ATP binding domain, and both transmembrane domains, could be partially working in the lung tissues.


Linkage of epidermolysis bullosa simplex to keratin gene loci. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder characterised by intraepidermal blistering of the skin. Two families with Weber-Cockayne EBS have been analysed for linkage to keratin gene loci. In the first family, linkage was found to chromosome 17 markers flanking the keratin 14 gene (D17S74: Zmax = +2.45, theta = 0.10; COL1A1: Zmax = +0.97, theta = 0.00) and markers near the keratin 5 gene on chromosome 12 were excluded (D12S17: Z less than -2.0, theta = 0.08; COL2A1: Z less than -2.0, theta = 0.13). In contrast, the second family showed linkage to the region containing the keratin 5 gene (D12S17: Zmax = +1.37, theta = 0.08; COL2A1: Zmax = +0.33, theta = 0.15) and was not linked to the keratin 14 gene (D17S74: Z less than -2.0, theta = 0.14). The Weber-Cockayne form of EBS is genetically heterogeneous with linkage to different keratin gene loci.


Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance. A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 5.7 residues per turn. Unique values for helical dimensions are also specified.


Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 A resolution. N-carbamoylsarcosine amidohydrolase from Arthrobacter sp., a tetramer of polypeptides with 264 amino acid residues each, has been crystallized and its structure solved and refined at 2.0 A resolution, to a crystallographic R-factor of 18.6%. The crystals employed in the analysis contain one tetramer of 116,000 M(r) in the asymmetric unit. The structure determination proceeded by multiple isomorphous replacement, followed by solvent-flattening and density averaging about the local diads within the tetramer. In the final refined model, the root-mean-square deviation from ideality is 0.01 A for bond distances and 2.7 degrees for bond angles. The asymmetric unit consists of 7853 protein atoms, 431 water molecules and four sulfate ions bound into the putative active site clefts in each subunit. One subunit contains a central six-stranded parallel beta-pleated sheet packed by helices on both sides. On one side, two helices face the solvent, while two of the helices on the other side are buried in the tight intersubunit contacts. The catalytic center of the enzyme, tentatively identified by inhibitor binding, is located at the interface between two subunits and involves residues from both. It is suggested that the nucleophilic group involved in hydrolysis of the substrate is the thiol group of Cys117 and a nucleophilic addition-elimination mechanism is proposed.


Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations. Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.


Behavior of postcapillary venule pericytes during postnatal angiogenesis. Autogeneic bone marrow was implanted into an artificially created cavity in a segment of rat sciatic nerve, after removal of nerve fascicles, without damaging the epineurium or surrounding microcirculation. Under these conditions, the bone marrow induces capillary growth and forms granulation tissue from surrounding tissues, the behavior of pericytes being studied in the preformed (preexisting) postcapillary venules of the latter. Beginning 20 h after bone marrow implantation, the pericytes of the preexisting postcapillary venules hypertrophy, with shortening of their processes, prominent nucleoli, dispersal of ribosomes into their free form, fragmentation of basal lamina, and increased DNA synthesis. The number of contact surfaces between pericytes and endothelium is noticeably lower than in controls. Many pericytes are in mitosis. Cells with a shape transitional between pericytes and interstitial fibroblast-like cells appear. In some cases, Monastral Blue (MB) was used as a marker of the cells in preexisting venule walls of the graft bed. In the earlier stages of the experiment, the MB labelling is restricted to the cytoplasm of pericytes and endothelial cells of postcapillary venules, and to the macrophages that occur in the space between pericytes and endothelium. Furthermore, the marker continues to be observed, at a later stage, in some of the following cells: pericytes and endothelial cells of the newly formed vessels, macrophages migrating into the interstitium, transitional cells between pericytes and fibroblasts, and typical fibroblasts of the granulation tissue. The present study provides greater evidence that preformed microvasculature pericytes are substantially activated during postnatal angiogenesis and granulation tissue formation, suggesting that they may contribute to the origin of new pericytes and fibroblasts.


[Detection of minimal residual disease using reverse transcriptase polymerase chain reaction technique]. Polymerase chain reaction (PCR) is a highly sensitive technique to detect minimal residual disease (MRD) of hematological malignancy by amplifying tumor specific nucleotide sequences. When breakpoint is located over large lesions of genomic DNA, like t(9;22) leukemia, PCR amplifying cDNA of chimeric mRNA, called reverse transcriptase PCR (RT-PCR), can be utilized. We applied this method for the detection of MRD in patients with t(1; 19) acute lymphoblastic leukemia. RT-PCR detection of MRD in patients with leukemia might be useful for estimating of the depth of remission, for disclosing preclinical relapse, and for evaluating the efficacy of in vitro purging in autologous bone marrow transplantation.


[Recent advances in the chemotherapy of leukemias]. Most recent progress in the treatment of leukemia and choice of therapies for obtaining "cure" from leukemia are discussed. Chemotherapy can now provide about 40% long term survival in acute myeloblastic leukemia (AML) and about 20% disease-free survival in acute lymphoblastic leukemia (ALL) of adults. Bone marrow transplantation (BMT) should be applied for the patients at risk in those leukemias (Cytogenetic abnormalities for AML and prognostic factors in ALL). In CML patients, BMT offers the only cure. Update result of interferon (IFN) therapy for CML is still a matter of controversy. Ex vivo treatment of autologous cells with IFN or drugs may be beneficial for CML patients without HLA identical donor.


[Recent progress in the chemotherapy program and its theoretical background--malignant lymphoma]. Recently developed third generation chemotherapy programs have improved long-term disease free survival of patients with non-Hodgkin's lymphoma of aggressive histology, as compared with their predecessors. These protocols have been developed based on the cancer chemotherapy principles derived mainly of experimental tumor studies by H. Skipper and his group, and the theoretical approach by Goldie and Coldman to the occurrence of and chemotherapeutic overcoming of resistant clones. They are characterized by the use of multiple non-cross-resistant drugs in maximally elevated dose intensity. Our third generation protocol, CAMBO-VIP, has produced 90% CR and 76% DFS at 3 years. Further improvement may be obtained by application of new drugs with different mechanism of action, effective means to destruct resistant cells, and further elevation of dose intensity by myelostimmulatory cytokines or transplantation of autologous/allogeneic peripheral blood or bone marrow stem cells.


[Differentiation therapy of hematologic malignancies: principles, current status and perspectives]. There is increasing evidence that various tumor cells can be induced by differentiation inducers including biological response modifiers, synthetic chemicals and conventional anticancer drugs to differentiate terminally both in vitro and in vivo into cells with normal characteristics. The differentiated cells lose their abilities for proliferation and transplantation in animals. Furthermore, survival times of the animals inoculated with various tumors were found to prolong by administration with the differentiation inducers. These basic findings suggest that induction of terminal tumor cell differentiation is another strategy for cancer therapy: differentiation therapy of cancer. Principles, current status and perspectives of the differentiation therapy of leukemia are reviewed in this article.


[High dose chemotherapy with autologous bone marrow transplantation and G-CSF for hematologic malignancies]. High dose chemotherapy (HDCT) followed by autologous bone marrow transplantation (ABMT) is one of the cure-oriented therapies for hematologic malignancies. Colony stimulating factors (CSFs) are currently used to stimulate hematopoiesis in various cytopenic conditions including post BMT. Granulocyte CSF (G-CSF) significantly accelerates granulocyte recovery after ABMT. Earlier recovery of neutrophils is associated with lower incidence of severe infections and shorter duration of reverse barrier nursing and hospitalization. Widespread use of G-CSF in ABMT may contribute to managing this treatment safely in multi-centers. Further prospective clinical study to define the optimal application and timing of HDCT with ABMT in hematologic malignancies is required.


[Diagnostic value of measurement of RNA in platelets by fluorescence flow cytometry]. We analyzed the RNA in platelets by fluorescence flow cytometry after staining with thiazole orange(TO) in whole blood samples from hematologically normal subjects and patients with thrombocytopenia. The percentage of TO-positive platelets and their mean fluorescence channel number in 32 control subjects were 6.2 +/- 2.5% (mean +/- SD) and 6.9 +/- 0.7, respectively. In 11 patients with idiopathic thrombocytopenic purpura, the percentage of fluorescently labeled platelets was significantly elevated (p less than 0.05) to 21.5 +/- 14.3%. By contrast, the proportion of positively stained platelets in 14 patients with thrombocytopenia due to impaired platelet production did not significantly differ from that of the controls, whereas the absolute counts of TO-positive platelets were significantly lowered (p less than 0.05). In both patient groups, the mean fluorescence channel numbers of TO-positive platelets were significantly elevated to 16.1 +/- 16.8 and 6.9 +/- 0.7, respectively (p less than 0.05, 0.005). We conclude that flow cytometric analysis of platelets after staining with TO provides information on the thrombopoietic activity in thrombocytopenic disorders. The main advantages of this method for clinical use are its simplicity and the rapidity.


Value of retroperitoneal lymph node dissection in advanced testicular seminoma. Seven patients with advanced seminoma of the testis (stages II C and III) were treated with 3 induction cycles of VAB-6 chemotherapy. Three patients had complete remission after chemotherapy and are alive disease free at 32, 38, and 40 months with no additional treatment. Four patients were subjected to retroperitoneal lymph node dissection for residual retroperitoneal masses measuring 2-4 cm after chemotherapy, which revealed fibrosis in 2 patients and metastatic seminoma in the other 2. Patients with metastatic residual masses were given postoperative radiation therapy to the retroperitoneum and are alive disease free at 33 and 34 months, while those with fibrosis are alive disease free at 30 and 52 months, respectively, with no additional treatment.


Retention of progenitor cell function in CD34+ cells purified using a novel O-sialoglycoprotease. We previously showed that the sialoglycoprotein, CD34, which is expressed on primitive human hematopoietic progenitor cells, is cleaved by a unique glycoprotease from Pasteurella haemolytica (P.h. glycoprotease). This proteolytic enzyme specifically cleaves glycoproteins rich in O-sialoglycans. Glycoproteins containing only N-linked glycans are not cleaved. Cleavage of the CD34 antigen results in the loss of epitopes detected by five of seven CD34-designated antibodies. In this study, we investigated the role of the P.h. glycoprotease in isolating CD34+ cells from unfractionated normal human bone marrow mononuclear cells (MNCs), and determined the effect of the glycoprotease on the proliferative capacity of the progenitor-enriched fraction. CD34+ cells were isolated from MNCs using immunomagnetic beads attached via a CD34 antibody whose epitope is susceptible to removal by the cleavage with the glycoprotease. Subsequent cleavage with P.h. glycoprotease for 30 min at 37 degrees C released the CD34+ cells from the beads with a recovery of up to 78%. Using a CD34 antibody whose epitope was not removed by the glycoprotease, up to 95% of the recovered cells expressed CD34. Compared to unseparated MNCs, the CD34+ cells showed the following enrichment of committed hematopoietic progenitors, as assayed in semi-solid media: CFU-GM, 45-fold; CFU-M, 13-fold; BFU-E, 26-fold and CFU-GEMM, 81-fold. Hematopoiesis was also studied in two-stage long-term bone marrow cultures in which the CD34+ cells were co-cultured over irradiated, allogeneic adherent layers. Output of CFU-GM over a seven week period from these cultures was similar to that from control cultures with autologous adherent-cell-depleted marrow MNCs. These data suggest that the loss of O-sialo-glycosylated peptide moieties from P.h. glycoprotease-released CD34+ cells neither affects the functional capacity of committed progenitors, nor impairs the proliferation of long-term culture-generating cells. The P.h. glycoprotease can be used to facilitate the isolation and recovery of functionally competent CD34+ cells at high yield and purity, without prior removal of other adherent cells. The ability to rapidly purify CD34+ cells using this non-cytotoxic enzyme has important implications for bone marrow transplantation as well as for gene transfer studies in vitro.


Effects of lorazepam tolerance and withdrawal on GABAA receptor operated chloride channels in mice selected for differences in ethanol withdrawal severity. Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) mice were treated with 5 mg/kg lorazepam for 7 days via implanted osmotic mini pumps. Following chronic drug treatment, brains were assayed for GABA-mediated chloride flux (GABA-Cl-). Under control (drug naive) conditions, brain membranes prepared from WSP and WSR lines did not differ in flunitrazepam or ethanol stimulation of GABA-mediated 36Cl- uptake, but the WSP lines were more sensitive to inhibition of 36Cl- flux by the inverse agonist, FG-7142. Membranes from lorazepam tolerant WSP and WSR mice were resistant to flunitrazepam- and ethanol-stimulation of GABA-Cl-. Withdrawal from chronic treatment, by an acute injection with the benzodiazepine antagonist RO15-1788, returned flunitrazepam stimulation of GABA-Cl- to near control levels in WSR membranes but not in WSP membranes and restored ethanol modulation of the channel to control levels in both lines. Inhibition of chloride flux by the benzodiazepine partial inverse agonist, FG-7142 was greater in membranes from WSP mice compared with WSR mice. Tolerance to lorazepam increased sensitivity of the WSR membranes to FG-7142 without altering the response in the WSP line. Again, withdrawal restored the Cl- flux response to FG-7142 back to near control levels. Lorazepam tolerance lowered [3H]-flunitrazepam binding affinity slightly only in the WSR strain with no change in binding number. Withdrawal from chronic lorazepam treatment produced no significant change in binding affinity or number. The initial genotypic differences in benzodiazepine inverse agonist sensitivity, may be related to the selection for withdrawal seizure severity. Chronic administration of lorazepam reduces the coupling between the benzodiazepine agonist site and the chloride channel and concomitantly increases coupling between the channel and the inverse agonist site, while withdrawal resets the receptor coupling back to control response levels. However, for the WSP line, this drug environment dependent shift in channel coupling bias appears to be deficient compared with the WSR line.


Mouse bone marrow micronucleus test using flow cytometry. At present, the micronucleus test is the most popular short-term assay for the in vivo detection of clastogens or spindle poisons. As micronuclei are rare events, it has been argued that the conventional microscopical analysis based on 500-2000 cells per animal does not provide enough sensitivity. In addition, the manual scoring of micronuclei is time-consuming and tedious. As an attempt to solve these problems, an automated method for the analysis of micronuclei in mouse bone marrow erythrocytes was developed using flow cytometry. Femoral bone marrow cells, from mice treated with known in vivo clastogens [mitomycin C (MMC): 0.5, 1 or 2 mg/kg body weight intraperitoneally; benzene: 1000, 2000 or 4000 mg/kg body weight orally] or vehicles for the clastogens (physiological saline, olive oil; for control animals), were fixed with 1% glutaraldehyde, suspended in 70% ethanol for storing, stained with 4',6-diamidino-2-phenylindole (0.5 microgram/ml) and analyzed (50,000 cells per sample) in an EPICS V flow cytometer using a Coherent 5W UV laser at 150-200 mV. The frequency of micronucleated erythrocytes (MNEs) was calculated from the cytometer, by a computer program involving model fitting to normal Gaussian distribution. Correlations between MNE frequencies obtained by manual microscopic observations (1000-2000 cells per animal) and by the flow cytometric measurements were good for both group results (means of 4-5 mice; linear correlation coefficient r = 0.89-0.998) and individual data (r = 0.82-0.96). Repeated experiments with MMC showed good reproducibility for the flow cytometric scoring of micronuclei.


Glutamine and macrophage function.

Glutamine and macrophage function. The effects of glutamine concentration on the phagocytosis of an opsonized antigen, the synthesis of RNA, and the production of interleukin-1 (IL-1) by macrophages were investigated in vitro. A minimum A minimum of 0.125 mmol/L glutamine was required for a significant increase in phagocytosis of opsonized sheep erythrocytes, compared with that recorded for macrophages cultured in the absence of glutamine. The synthesis of 3H-RNA by macrophages also required 0.125 mmol/L glutamine in the culture medium before it was significantly increased above the levels of control cultures. A minimum of 0.03 mmol/L glutamine was required for the induction of significant levels of IL-1 by lipopolysaccharide (LPS)-stimulated macrophages. Therefore, recent findings suggesting that decreases in plasma glutamine resulting from major burn injury, sepsis, trauma, and surgery may be partly responsible for the associated impairment of immune function now have a basis in both phagocytosis and in modulation of the synthesis of IL-1 (the first cytokine of the interleukin cascade that leads to specific immunity) by macrophages, in addition to the previously established dependency of lymphocytes on external sources of glutamine for their replication.


[Therapy of malignant liver tumors in childhood. An intermediate report of the HB-89 multicenter study of the GPOH]. After a pilot phase in 1988 the liver tumour study HB-89 of the German Society of Pediatric Oncology and Hematology (GPOH) was started on January 1st 1989. It comprises the malignant epithelial liver tumours of childhood--hepatoblastoma and hepatocellular carcinoma. The objectives of the study are 1. to reduce surgical complications and incomplete tumour resections by restricting the primary operation to a lobectomy of the liver in case of small tumours or to a biopsy in case of extended disease, and 2. to reduce the size of large tumours by means of chemotherapy for subsequent resection. The tumourreductive and adjuvant chemotherapy consists of the combinations of ifosfamide, cisplatinum, adriamycin (IPA) and cisplatinum plus adriamycin as continuous infusion (PA-cont). Up to December 31st 1991 a total of 78 pediatric liver tumours were registered, 51 of those hepatoblastomas and 10 hepatocellular carcinomas. 19 (37%) hepatoblastomas were resected at primary operation (stage I and II). After chemotherapy 24 (83%) of 29 primarily inoperable hepatoblastomas (stage III and IV) became resectable, one child received a liver transplant. At the time all hepatoblastoma patients of stages I and II and 20 (69%) of stages III and IV are in remission, the overall remission rate is 81% (39/48). Seven patients with multifocal hepatoblastoma had an unfavourable outcome, only two are free of tumour. Of 10 patients with a hepatocellular carcinoma only three with a primary complete tumour resection are disease free. The IPA and PA-cont therapies were highly effective on hepatoblastomas (96%), but not on hepatocellular carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)


Rapid screening for deletion mutations in the hprt gene using the polymerase chain reaction: X-ray and alpha-particle mutant spectra. Conditions were devised for the isolation of DNA from single-mutant colonies on dishes, to give reproducible results in the polymerase chain reaction (PCR). Primers for 3 exons of the hamster hprt gene were used in a multiplex reaction to show rapidly whether the mutants carried deletions at these sites. 138 independent mutants were screened in total, some spontaneous and others induced by X-rays or by alpha-particles from plutonium-238. Few deletions were found among the spontaneous set, while 'total' gene deletions formed about half the mutants found after irradiation. At equitoxic doses, little difference in mutant spectrum was found for the X-ray set compared to the alpha-particle set. This rapid technique should be applicable to many instances of comparative mutagenesis.


Telomere association of human chromosomes induced by aphidicolin. Telomere associations were studied in metaphase chromosomes from 96-h cultures of peripheral blood lymphocytes of two healthy women, treated with 0.4 microM aphidicolin for the last 72 h. Telomere associations were encountered in 2.9% and 3.2% of the metaphases screened, whereas no such associations were encountered in 5-fluorodeoxyuridine-treated cultures. The chromosome arms involved in telomere associations were nonrandom: 1q, 2q, 3q, 6p and 16q were more frequently involved in the associations (P less than 0.01). Of the 51 combinations of telomere associations encountered, those occurring nonrandomly were 1q/2q, 2q/2q, 4q/4q, 6q/6q and 6p/6p associations.


Effects of antioxidants on induction of micronuclei in rat peripheral blood reticulocytes by potassium bromate. Micronucleus induction in male F344 rat peripheral blood by potassium bromate (KBrO3), a rat renal carcinogen, and its inhibition by several antioxidants were studied using the acridine orange supravital staining method. The frequency of micronucleated reticulocytes (MNRETs) peaked 32 h after a single i.p. treatment of rats with KBrO3 at a dose of 60 mg/kg. Co-treatment with glutathione (GSH) or cysteine (Cys) i.p. at doses of 800 mg/kg and 400 mg/kg, respectively, 30 min before and 30 min after the KBrO3 treatment significantly inhibited the micronucleus induction by KBrO3. Daily i.g. administration of vitamin C for 5 days at a dose of 200 mg/kg/day was also effective in protecting against micronucleus induction by KBrO3 given on the 4th day. However, co-treatment with superoxide dismutase in liposome-encapsulated form by i.p. injection at a dose of 18,000 U/kg 30 min before and 30 min after the KBrO3 application exerted no effect. The results indicate that antioxidants, especially sulfhydryl compounds, have protective potential against the clastogenicity of KBrO3, also suggesting that active oxygen species may play an important role in its clastogenicity.


Persistence of chromosomal lesions induced in actively proliferating bone marrow cells of the mouse. The persistence of chromosomal lesions induced in vivo by mitomycin C (MMC) was evaluated by cytogenetic analysis of mouse bone marrow cells. Chromosome aberration (CA) and micronucleus (MN) frequencies were estimated at different times after treatment, up to 42 days. The frequency of CA per cell decreased in the first 3 days after treatment, but a secondary peak appeared on the 4th day, followed by a stabilization around 0.03 CA per cell (significantly different from the control value), which persisted up to 17 days. At the next time intervals tested (28 and 42 days), the CA frequency returned to the control level. In disagreement with these data obtained directly on metaphases, the MN frequency, as evaluated in polychromatic erythrocytes, decreased quickly after treatment, reaching the control value on the 5th day. We attempted to enhance the sensitivity of the MN test by using CREST antibodies and indirect immunofluorescence. However, higher proportions of CREST- MN in treated than in control animals were observed only at short time intervals, confirming the results obtained with the conventional MN assay.


Strand specificity for mutations induced by (+)-anti BPDE in the hprt gene in human T-lymphocytes. Mutations in the hprt gene in T-lymphocyte clones isolated from primary cultures treated with the (+)-anti enantiomer of 7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE) in vitro, and from untreated control cultures, were characterized using polymerase chain reaction and direct sequencing of hprt cDNA and genomic fragments. The spectrum of BPDE-induced mutations was very specific and clearly different from the background spectrum, which comprised many different types of mutations. Of the BPDE-induced mutations, 20/22 were transversions of GC base pairs and 18/22 were GC greater than TA transversions, which is in agreement with what has been found in other mammalian systems. While no particular 'hotspot' was observed for BPDE in the hprt gene, a sequence context specificity was detected. Ten of the 14 BPDE-induced mutations in the coding region were located in the sequence context AGG, and 2 in AG dinucleotides, which indicates that such sequences are sensitive to BPDE mutagenesis. Nine of the 22 BPDE-induced mutations and 2/12 background point mutations caused mRNA splicing errors. Six of the BPDE-induced splicing errors were caused by GC greater than TA transversions in the AG dinucleotide of different splice acceptor sites, which indicates that these sites may be frequent targets of BPDE mutagenesis. All mutated GC base pairs in the BPDE-induced spectrum were oriented so that the guanine was located on the non-transcribed strand. Assuming that the premutagenic lesion in these cases was covalent binding of BPDE to guanine and that BPDE bound randomly to both strands, the strand specificity of the BPDE-induced mutations indicates that preferential excision repair of BPDE adducts on the transcribed strand occurs in the hprt gene in human T-cells.


Forward mutations and DNA-protein crosslinks induced by ammonium metavanadate in cultured mammalian cells. Ammonium metavanadate yielded a dose-dependent increase in mutation frequency at the V79 hprt locus following a 24-h exposure period in serum-free F12 medium. Vanadate also increased the mutation frequency of V79 cells by exposure of cells in salts-glucose medium, but these effects were not as striking, or as dose-dependent as they were in serum-free F12 medium. Ammonium metavanadate enhanced the mutation frequency in a V79 variant containing a transfected bacterial gpt gene. These cells are known to be more responsive to oxidative type mutations, and to mutations involving deletions. Although the absolute level of mutations was greater in these cells with ammonium metavanadate, so was the background, and these cells did not exhibit an enhanced mutagenic response to vanadate when compared to the wild-type V79 cells. The vanadate results were compared to a positive control potassium chromate, which exhibited a dose-dependent increase in mutation frequency. Ammonium metavanadate induced DNA-protein crosslinks formation in both Chinese hamster ovary and human MOLT4 cells, and the role of these relatively unrepaired genetic lesions in the mutations produced by vanadate and chromate are discussed.


X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. XI. Heterozygous effects of gene/point mutations of genotype ad-3A or ad-3B. Previous studies on X-ray-induced irreparable adenine-3 mutants (designated ad-3IR), induced in heterokaryon 12 of Neurospora crassa, showed that they were not recessive, and that they demonstrated heterozygous effects in terms of markedly reduced linear growth rates as compared with a wild-type control (de Serres, 1965, 1988). Homology tests on X-ray-induced irreparable mutants showed that they map, in the main part, as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential, function) between ad-3A and ad-3B (de Serres, 1969, 1989a). Studies on a larger sample of X-ray-induced multilocus deletion mutations of genotype (ad-3A)IR or (ad-3B)IR (de Serres et al., 1992) demonstrated that heterozygous effects are allele specific and that there was no correlation with genotype, radiation dose or complementation map position. Furthermore, the heterozygous effects of multilocus deletions in the ad-3 region can be modified genetically and biochemically (de Serres and Miller, 1988). In the present paper, the heterozygous effects of X-ray-induced gene/point mutations of genotype ad-3AR or ad-3BR, induced in heterokaryon 12 (Webber and de Serres, 1965; de Serres, 1988, 1989a), were determined. The studies presented in this paper show that 8.1% (3/37) of X-ray-induced ad-3AR mutations exhibit heterozygous effects in terms of reduced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus, and 10.8% (4/37) in forced dikaryons with a multilocus deletion mutation covering the ad-3B locus. In addition, 24.3% (9/37) of ad-3AR mutations exhibit heterozygous effects in terms of enhanced linear growth rates in forced dikaryons with a gene/point mutant at the ad-3B locus. Similar studies with X-ray-induced ad-3BR mutations showed that 54.9% (28/51) exhibit heterozygous effects in terms of reduced growth rates in forced dikaryons with a gene/point mutant at the ad-3A locus and 100.0% (48/48) in forced dikaryons with a multilocus deletion covering the ad-3A locus. These studies have also shown that about a 13-fold higher percentage of X-ray-induced multiple-locus mutations of genotype ad-3AR + RLCL have heterozygous effects resulting in reduced growth rates than X-ray-induced single-locus mutations of genotype ad-3AR. The overall data base on X-ray-induced ad-3 gene/point mutations in the present studies demonstrates that heterozygous effects are allele specific, genotype specific, and locus specific.(ABSTRACT TRUNCATED AT 400 WORDS)


Directly acting geno- and cytotoxic agents from a wild mushroom Dermocybe sanguinea. The wild mushroom, Dermocybe sanguinea, contains several anthraquinone pigments, of which emodin (1,3,8-trihydroxy-6-methylanthraquinone) is quantitatively the most important. In our preliminary tests, Dermocybe sanguinea extracts were genotoxic without metabolic activation. The ethanol extract of Dermocybe sanguinea was fractionated by flash chromatography, and the emodin contents of the fractions were determined by HPLC. Their genotoxicities were assayed using a bacterial repair assay and sister-chromatid exchange analysis. The cytotoxicity of the fractions was assayed with mouse hepatoma cells using growth inhibition as the endpoint. The results of the biological tests were compared with those obtained with pure emodin. It was concluded that, in addition to emodin, Dermocybe sanguinea contains several other geno- and cytotoxic compounds.


Lower birth weight of offspring born after self-poisoning of parent. Birth weight was evaluated in 777 and 217 livebirths, respectively, of index females and female partners of index males born before and after severe self-poisoning with drugs. Birth weight was also evaluated in matched controls. Babies born to index females months or years after an attempted self-poisoning were found to have a lower birth weight than before this suicide attempt. The difference in the birth weight of subsequent pregnancies of index and control females was also highly significant. A similar trend was observed in livebirths of female partners of index males. However, the differences were not significant between previous and subsequent pregnancies and between index cases and matched controls.