A comparison of electrophysiologically guided antiarrhythmic drug therapy with beta-blocker therapy in patients with symptomatic, sustained ventricular tachyarrhythmias.

Antiarrhythmic drug therapy guided by invasive electrophysiologic testing is now widely used in patients with symptomatic, sustained ventricular tachyarrhythmias. We conducted a prospective, randomized trial in 170 patients to investigate whether this approach would improve long-term outcome. Patients whose arrhythmia was inducible by programmed electrical stimulation were assigned to treatment with electrophysiologically guided drug therapy based on serial testing (61 patients) or with metoprolol (54 patients). Electrophysiologically guided therapy consisted of serial testing of antiarrhythmic agents to identify the first one that rendered the arrhythmia noninducible. The 55 patients whose arrhythmia was noninducible during the initial electrophysiologic test were also treated with metoprolol. During a mean (+/- SD) follow-up period of 23 +/- 17 months, recurrent, nonfatal arrhythmia occurred in 44 patients and sudden death due to cardiac factors in 27. The incidence of symptomatic arrhythmia and sudden death combined was virtually the same in the two groups with inducible arrhythmia after two years of observation (electrophysiologically guided therapy vs. metoprolol therapy, 46 percent vs. 48 percent). The outcome was more favorable in the patients with noninducible arrhythmia at base line (75 percent had neither adverse event) than in those with inducible arrhythmia who were assigned to metoprolol therapy (P = 0.009 by log-rank test). Only 6 of the 29 patients (21 percent) with inducible arrhythmia that became noninducible during drug therapy had recurrent arrhythmia or sudden death, as compared with 21 of the 32 patients (66 percent) with arrhythmia that continued to be inducible (P less than 0.001). A multivariate regression analysis identified continued inducibility of the arrhythmia as an independent predictor of recurrent arrhythmia or sudden death (relative risk, 7.3; 95 percent confidence interval, 2.3 to 23.2; P less than 0.001). As compared with metoprolol therapy, electrophysiologically guided antiarrhythmic drug therapy did not improve the overall outcome of patients with sustained ventricular tachyarrhythmias. However, effective suppression of inducible arrhythmia by antiarrhythmic drugs was associated with a better outcome than was lack of suppression.


Supraspinal administration of [D-Met2]FMRFamide produces a naloxone-sensitive increase in heart rate in unrestrained spontaneously hypertensive rats.

Intracerebroventricular (i.c.v.) administration of either Phe-Met-Arg-Phe-NH2 (FMRFamide; molluscan cardioexcitatory neuropeptide; 3-30 micrograms) or the FMRFamide analog Phe-D-Met-Arg-Phe-NH2 ([D-Met2]FMRFamide; 15 micrograms) to conscious unrestrained spontaneously hypertensive rats (SHR) produced a relatively long lasting (greater than 1 h) increase in heart rate. The increase in heart rate produced by [D-Met2]FMRFamide was attenuated by i.c.v. injection of the opiate antagonist naloxone (2 micrograms). These results extend to a second endpoint an apparent opioid agonist-like (naloxone-reversible) action of [D-Met2]FMRFamide.


Human cytomegalovirus in rejected kidney grafts; detection by polymerase chain reaction.

Human cytomegalovirus (CMV) infections are frequently associated with graft rejection in the immunosuppressed patients following organ transplantation. Thirty-four tissue samples from rejected kidneys and 18 samples from normal adult kidneys obtained from autopsies were investigated for the presence of CMV-DNA by the polymerase chain reaction (PCR) and by immunohistochemistry. DNA extracted from renal tissues after proteinase K digestion was specifically amplified in 32 cycles using primers which flank a 147 bp DNA fragment of the immediate early CMV gene and analysed by slot-blot hybridization with digoxigenin-labelled detection oligonucleotides. CMV-DNA was detected by PCR in a range from 0.1 fg up to 100 fg in 14 (41%) rejected kidney transplants. Comparative immunohistological analysis revealed presence of CMV in only three biopsies of these rejected kidneys. Furthermore, CMV-DNA was also found in four of 18 (22%) normal donor kidneys. These results reveal that CMV is often present in rejected kidneys and that the infection can be transferred from the donor to the recipient, since the normal adult kidney appears to be a frequent site of latency for CMV. No differences in local immunological changes, characterized by interstitial mononuclear leukocyte infiltration as well as by aberrant expression of HLA-class II antigens and of ICAM1 on proximal tubular epithelial cells, could be detected by further immunohistological analysis between grafted kidneys at late stage of rejection with and without CMV infection.


Comparative evaluation of the neuromuscular and cardiovascular effects of pipecuronium, pancuronium, atracurium, and vecuronium under isoflurane anesthesia.

The neuromuscular and cardiovascular effects of intubating doses of pipecuronium 80 micrograms/kg, pancuronium 100 micrograms/kg, atracurium 500 micrograms/kg, and vecuronium 100 micrograms/kg were compared in 62 patients under isoflurane (end-tidal concentration = 0.5-1%) anesthesia. Pipecuronium, pancuronium, and vecuronium had no significant effect on systolic or diastolic blood pressure. In one patient the administration of atracurium resulted in significant hypotension. Heart rate was significantly increased only after the administration of pancuronium. The neuromuscular-blocking effect of pipecuronium and pancuronium appears to be twice as long as that of vecuronium and atracurium. Administration of neostigmine resulted in significantly faster recovery of muscle function in patients receiving vecuronium or atracurium. Although pipecuronium's neuromuscular-blocking effect is similar to that of pancuronium, its lack of cardiovascular effects more closely resembles that of vecuronium.


The post-myocardial infarction patient with hypertension. Long-term outcome.

Hypertension increases the risk of reinfarction and sudden death in post-myocardial infarction (MI) patients. The same s true of coexisting left ventricular hypertrophy. Fortunately, these two risk factors may be modified by pharmacotherapy. In this article, Dr Boden describes the complementary use of beta blockers and selected calcium channel blockers for secondary prevention after acute MI, particularly in the subset of patients who also have hypertension.


Identification of a transcriptional repressor down-regulated during preadipocyte differentiation.

During differentiation of 3T3-L1 preadipocytes into adipocytes, the transcription of adipocyte genes, including the stearoyl-CoA desaturase 2 (SCD2) gene, is activated. Transfection experiments with chimeric SCD2 promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs revealed a preadipocyte repressor element (PRE) capable of repressing transcription of the reporter gene in preadipocytes but not in adipocytes. DNase I protection and gel retardation analyses were used to localize the PRE site between nucleotides -435 and -410 of the SCD2 promoter and to identify a nuclear PRE binding protein present at high levels in preadipocytes and HeLa cells but lacking or inactive in adipocytes. Southwestern blot analysis indicated that the PRE binding protein has an apparent molecular mass of approximately 58 kDa. A single copy of the PRE site, inserted upstream of the simian virus 40 enhancer/promoter of pSV2CAT, was capable of strongly repressing transcription of the reporter gene in preadipocytes and HeLa cells but not in adipocytes. Taken together these results suggest that the PRE site and binding protein may regulate transcription of SCD2 and possibly other adipocyte genes by inhibiting their transcription in preadipocytes.


Superantigen staphylococcal enterotoxin B-induced T-helper cell activation is independent of CD4 molecules and phosphatidylinositol hydrolysis.

The role of the CD4 molecule in activation of T-helper cells was examined by investigating the effect of an anti-CD4 monoclonal antibody (Leu3a) in conventional peptide antigen-specific cloned T-helper cells that are also reactive to staphylococcal enterotoxin B (SEB). These T-helper cell clones are CD4+/CD45RO+/T-cell antigen receptor beta-chain variable region 12-positive and can respond to nominal peptide antigens and SEB by proliferation in the presence of class II major histocompatibility complex-expressing accessory cells. Although antigen and SEB were comparable in their ability to induce proliferative responses, interleukin 2 (IL-2) production, and IL-2 receptor alpha-chain expression, stimulation with SEB failed to trigger phosphatidylinositol hydrolysis or a rise in the intracellular free calcium ion concentration. Leu3a treatment inhibited antigen-induced proliferative responses of T cells with concomitant suppression of IL-2 production and IL-2 receptor expression. In contrast, SEB-induced responses were unaffected by Leu3a. These findings indicate that the functional consequences of binding (ligation) of conventional antigen and of superantigen with the T-cell receptor are distinct in the context of both signal transduction pathways and participation of CD4 molecules.


Characterization of a metabotropic glutamate receptor: direct negative coupling to adenylyl cyclase and involvement of a pertussis toxin-sensitive G protein.

We have characterized a G-protein-coupled glutamate receptor in primary cultures of striatal neurons. Glutamate, quisqualate, or trans-1-aminocyclopentane-1,3-dicarboxylate inhibited by 30-40% either forskolin-stimulated cAMP production in intact cells or forskolin plus vasoactive intestinal peptide-activated adenylyl cyclase assayed in neuronal membrane preparations. These inhibitory effects were suppressed after treatment of striatal neurons with Bordetella pertussis toxin, suggesting the involvement of a heterotrimeric guanine nucleotide-binding protein (G protein) of the G(i)/G(o) subtype. The pharmacological profile of this glutamate receptor negatively coupled to adenylyl cyclase was different from that of the metabotropic Qp glutamate receptor coupled to phospholipase C in striatal neurons and from that of the recently cloned "mGluR2" glutamate receptor, which is negatively coupled to adenylyl cyclase when expressed in non-neuronal cells.


Expression pattern of a murine homeobox gene, Dbx, displays extreme spatial restriction in embryonic forebrain and spinal cord.

Homeobox genes specify regional identity during development. A homeobox sequence that we have named Dbx was isolated from 13.5-day embryonic mouse telencephalon cDNA. The Dbx homeodomain shows highest sequence homology to Drosophila H2.0 and chicken CHox E. We report here the expression pattern of Dbx during mouse embryogenesis. In situ hybridization analyses indicate that Dbx is expressed exclusively within the embryonic central nervous system in a highly restricted manner. Dbx transcripts are detected within a region of the prospective cerebral cortex of the midgestation telencephalon. Dbx is also expressed in the diencephalon as well as in two thin continuous columns of neuroblasts within the hindbrain and spinal cord. This expression is limited to regions of active mitosis. Dbx may act to specify subsets of neuroblasts during the development of the central nervous system.


Endocytosis by antigen presenting cells: dendritic cells are as endocytically active as other antigen presenting cells.

Although dendritic cells are the most potent of all antigen presenting cells, they have paradoxically been regarded as having only a minimal capacity for endocytosis, which is a crucial step in antigen processing prior to presentation. Previous studies of dendritic cells, which are only available in small numbers, have been restricted to measurement of long-term endocytosis and so have stressed lysosomal accumulation. Measurement of traffic through late endosomes, which are closely related to the organelle in which antigen processing occurs, has, to date, required large numbers of cells and therefore has not been possible for dendritic cells. To resolve the paradox for dendritic cells, we have developed a flow cytometric assay of fluid-phase endocytosis that assesses late endosomal traffic by kinetic analysis of exocytosis in small numbers of cells. Using this assay, we show that fluid-phase endocytosis--in particular, traffic through late endosomes--is as active in dendritic cells as in other antigen presenting cells.


3'-Azido-3'-deoxythymidine-induced reduction in the ability of uninfected CD4-expressing cells to participate in syncytium formation.

A cocultivation assay system consisting of uninfected human T cells and cells chronically infected with human immunodeficiency virus type 1 has been used to investigate syncytium formation in short-term assays. Continuous treatment or short-term pretreatment of uninfected CD4-expressing human T-cell lines with 3'-azido-3'-deoxythymidine (AZT) reduces the ability of these cells to participate in syncytium formation when mixed with chronically infected cells. The effect of AZT on syncytium formation is observed both as a reduction in the number of syncytia and as a reduction in the size of the syncytia that are detected. This syncytium-reducing effect of AZT is dose and time dependent and does not result from a modulation of CD4 antigen expression on the cell surface of uninfected, treated cells. Maximum syncytium reduction is observed with the continuous presence of AZT; however, pretreatment for times as short as 15 min results in a significant reduction in syncytium formation. Since reverse transcription is not required for efficient syncytium formation, the syncytium-reducing effect of AZT on uninfected human cells may represent an antiviral property of AZT with important therapeutic potential.


Qualitative shift of lymphokine production in response to stimulation, as a consequence of preactivation in vivo or in vitro.

Lymphokine production, analysed at the single cell level, was compared in resting and primed T-cell populations. Cells were preactivated in vitro by repeated mitogen stimulations, or isolated as large, low density cells naturally activated in vivo, from normal spleens of unimmunized animals. A similar qualitative shift in the pattern of lymphokines synthesized after restimulation was found as a result of in vivo and in vitro preactivation of cells. Repeated stimulations in vitro resulted in a qualitative shift in the lymphokines produced in response to activation, from a dominance of IL-2 during the first and second culture, to a dominance of IL-4 and IL-5 in the later stimulations. In vivo activation lead to a similar separation of lymphokine production as primarily IL-2 was made by small resting cells, while large cells preferentially produced IL-4 and IL-5. IFN-gamma was produced by both small and large cells. Preactivation in vitro lead to a more rapid appearance of lymphokines during restimulation. In contrast, the in vivo naturally activated cells responded with a slow onset of lymphokine production when stimulated in vitro.


Activation of HLA-DR and interleukin-6 gene transcription in resting T cells via the CD2 molecule: relevance to chronic immune-mediated inflammation.

Only a minority of T cells at cell-mediated immune lesions are antigen specific. In the lesions of human autoimmune disease, such as the synovial membrane in rheumatoid arthritis, the T cells are activated as shown by a variety of phenotypic and functional changes including the expression of HLA-DR and the production of interleukin-6 (IL-6). The stimulatory pathway involved is unknown but does not seem to involve the T-cell receptor. Alternative pathways of activation which may be involved include the CD2 molecule. It is shown that the formation of sheep red blood cell (SRBC) rosettes with resting T cells from human peripheral blood, which is equivalent to CD2/LFA-3 binding, leads to the de novo transcription of the HLA-DR and IL-6 genes and the expression of HLA-DR on the surface of the T cells. There was no transcription of the interleukin-2 (IL-2) or the interleukin-2 receptor (IL-2R) genes and Tac expression was not seen. The rosetted T cells did not proliferate. These are all characteristics of T cells at chronic inflammatory sites. It is concluded that receptor-ligand interactions between CD2/LFA-3, which are expressed in increased amounts in the rheumatoid joint, may be one pathway by which antigen non-specific T cells are recruited as effector cells in lesions of human autoimmune disease.


Antibodies to eukaryotic, including autologous, native DNA are produced during BK virus infection, but not after immunization with non-infectious BK DNA.

The contemporary view concerning the origin of anti-dsDNA antibodies is that eukaryotic dsDNA is not immunogenic. Results presented here, however, show (1) that inoculation of rabbits with BK virus elicits antibodies to eukaryotic, including autologous, dsDNA, (2) that the transition from a non-immunogenic to an immunogenic state of autologous dsDNA depends on productive infection with BK virus, and (3) that inoculation with protein-free circular BK dsDNA initiates both infection in vivo and production of antibodies to autologous dsDNA. Non-infectious linearized BK dsDNA did not elicit any anti-dsDNA antibodies, while the same DNA molecule, when complexed with methylated bovine serum albumin, elicited anti-dsDNA antibodies solely recognizing BK dsDNA. Neither of the two linearized BK dsDNA preparations initiated infection. Using two different techniques, we could demonstrate that two separate sets of anti-dsDNA antibodies were produced during viral infection; one recognizing BK dsDNA, and the other recognizing autologous dsDNA. Thus, in contrast to previous assumptions, autologous dsDNA may be immunogenic. Based on the present results, we propose that autologous dsDNA can be rendered immunogenic through complex formation with viral DNA binding protein(s) such as the structural protein VP1 or the tumour antigen T. Such DNA-protein complexes may bypass a putative T-cell tolerance to autologous dsDNA.


Direct nucleotide sequencing using total cellular RNA from dengue-virus-infected mosquito cells.

In recent years, a large amount of nucleotide sequence data for dengue viruses has been published. Most of it was derived by sequencing cDNA synthesized from highly purified genomic viral RNA. This paper presents a simple and rapid method for the isolation of total RNA from mosquito cells infected with dengue viruses. This RNA can be used for direct nucleotide sequencing with specific primers without the need for further purification.


A study of nephropathia epidemica among military personnel in Sweden.

The incidence of nephropathia epidemica (NE), caused by Puumala virus, among military troops operating in an endemic area of Sweden was investigated. A total of 705 soldiers (age 18-25) involved in field training in three different endemic counties were bled twice within a 6-month interval. Three individuals seroconverted when tested for Puumala virus antibodies by IgG ELISA. Symptoms typical of NE were not recorded in any of the patients. However, mild febrile episodes were recorded in 2 of the 3 individuals. One serologically confirmed soldier with typical NE symptoms was found in another group of 12,000 troops. This soldier fell ill 3 weeks after a 7-day-long field exercise. The present study indicates that military populations are at considerably greater risk of contracting NE when compared to the entire population residing in the same area.


Treatment strategies for daily life silent myocardial ischemia: a correlation with potential pathogenic mechanisms.

Recent investigations of SMI occurring during daily life have advanced our understanding of the pathophysiology of myocardial ischemia. These contributions have directed our attention away from "chest pain" alone and physical exertion as the central provoking factor toward transient myocardial ischemia and its broader triggers and consequences. Transient myocardial ischemic episodes, the majority of which are silent, are found in a subset of patients with any clinical manifestations of CAD (eg, stable angina, unstable angina, myocardial infarction, and sudden death), as well as in those patients with CAD who are and have been totally asymptomatic. These episodes are an independent predictor of increased risk for future cardiac events. Most medical therapy and revascularization therapies have the potential to prevent or relieve these silent episodes; however, we do not yet know which method is superior in reducing SMI episodes or preventing future cardiac events. Furthermore, the benefit of reducing SMI versus the cost and potential morbidity of these chosen therapies is not known. At least three trials are now underway to examine some of these concerns (Table 2). Focus on pain relief alone does not appear to be an adequate approach to alter outcome in patients with CAD and may prove insufficient to control SMI. Until these issues are resolved, we believe a conservative approach to the management of patients with CAD is warranted. Documentation of ischemia (painful or painless) is essential. Three general principles should be kept in mind. First, the presence of detectable ischemia is of central importance. This information should be used in the overall risk assessment of the patient. Second, the level of concern or aggressiveness of treatment should be based on the risk associated with the ischemic abnormalities documented (Table 3). The exercise stress test is the most useful to begin this process. The detection of ischemic-type ST-segment depression, either silent or painful, at a low workload (eg, less than or equal to 120 beats per minute or less than or equal to 6.5 metabolic equivalents [METS]) implies high risk for adverse outcome. Likewise, these ST-segment changes occurring in leads that reflect multiple coronary artery distribution, of greater than 2 mm in magnitude and persisting for greater than 6 minutes, are all markers for high risk. Thallium redistribution defects occurring at low work loads, in multiple areas, associated with increased lung uptake and enlargement of the cardiac pool all imply high risk.(ABSTRACT TRUNCATED AT 400 WORDS)


Protein oxidation and aging.

A number of systems that generate oxygen free radicals catalyze the oxidative modification of proteins. Such modifications mark enzymes for degradation by cytosolic neutral alkaline proteases. Protein oxidation contributes to the pool of damaged enzymes, which increases in size during aging and in various pathological states. The age-related increase in amounts of oxidized protein may reflect the age-dependent accumulation of unrepaired DNA damage that, in a random manner, affects the concentrations or activities of numerous factors that govern the rates of protein oxidation and the degradation of oxidized protein.


Acute resistant asthma caused by excessive beta-2-adrenoceptor agonist inhalation and reversed by inhalation of beclomethasone.

With the aim of devising an improved beta 2-agonist treatment regimen for patients presenting with acute severe resistant asthma, a double-blind, placebo-controlled randomised study of the effect of fenoterol and beclomethasone inhalations was done in 40 patients. Fenoterol inhalations had minimal effect in acute resistant asthma, but significant improvement was obtained when beclomethasone inhalations were given before fenoterol inhalations. Sequential beclomethasone and fenoterol inhalations can therefore be used as a preliminary emergency treatment for these patients.


Restriction fragment length polymorphism of the apoprotein A-I-C-III gene cluster in control and stroke-prone white and black subjects: racial differences.

The presence of known restriction fragment length polymorphisms in the apoprotein A-I-C-III gene cluster, which encodes their respective apoproteins, was investigated using the restriction enzymes Sac I and Pst I to determine the potential role of genetic variations for stroke risk in an American population. Ninety-eight subjects (70 white, 28 black subjects), both normal controls with no carotid stenosis and those with carotid stenosis believed at risk for stroke, defined as showing stenosis focally or diffusely at that site, composed the study population. Sac I polymorphic S2 allele frequency was higher in stroke-risk groups, whereas Pst I polymorphic P2 allele frequency was similar in control and stroke-risk groups. Significantly higher levels of serum cholesterol, triglycerides, and low density lipoprotein (p less than 0.05) and significantly lower levels of high density lipoprotein (p less than 0.05) were observed in stroke-risk groups with diffuse stenosis. Results of our study with the two racial groups show the following: the frequency of Sac I polymorphism was significantly higher in American black compared with American white subjects (chi 2 = 3.92, p less than 0.05). Among serum lipids, triglycerides were significantly higher in white compared with black subjects (p less than 0.05). In white subjects, carotid artery stenosis was associated with significantly elevated total cholesterol and low density lipoprotein (p less than 0.01) but not with Sac I polymorphism. In black subjects the converse was observed, namely, the Sac I polymorphic S2 allele seemed to be associated with carotid bifurcation stenosis but did not reach statistical significance because of the small number of subjects. In addition, Sac I polymorphism did not correlate with any lipid profile. Pst I polymorphism was not associated with any lipid profile or carotid artery stenosis abnormalities. Our results indicate that carotid artery stenosis identifies white subjects with increased plasma total cholesterol and low density lipoprotein, an atherogenic profile, but not with Sac I polymorphism. These findings suggest that carotid bifurcation stenosis in white subjects is associated with an atherogenic lipid profile but not with apoprotein A-I-C-III restriction fragment length polymorphisms. In black subjects, Sac I polymorphism seems to identify those individuals with significant carotid stenosis, a necessary precursor to atherothrombotic brain infarction, but not those with elevated total cholesterol, elevated low density lipoprotein, and/or reduced high density lipoprotein. These results suggest that Sac I polymorphism may identify black subjects at increased risk for atherothrombotic brain infarctions.


Fibrin glue from single-donation autologous plasmapheresis.

A method is described for preparing fibrin glue from fibrinogen obtained by double cryoprecipitation of plasma collected by plasmapheresis. Plasma (average volume, 528.8 +/- 86 mL), collected from 12 plasmapheresis donors was cryoprecipitated twice. When the cryoprecipitated pellet was resuspended in 0.5 to 2 mL of saline (total volume of saline-resuspended fibrinogen: 5 mL), the average yield of fibrinogen was 78.4 +/- 18.3 mg per mL. This is comparable to commercial preparations, is suitable for clinical use, and offers greater safety at a reduced cost.


Analysis of the gene polymorphism of ABO blood group specific transferases helps diagnosis of acquired B status.

Blood group typing of an aged patient suffering from ileus provided evidence for an acquired B. As a parameter independent of cell membrane molecules or secreted blood group substances, the nucleotide sequence polymorphism of A and B transferases was investigated. Restriction fragment length polymorphism of DNA chains amplified in a polymerase chain reaction from the coding region of the glycosyltransferase indicated that no gene for B transferase was present in the patient's genome. We conclude that the assessment of polymorphism of AB0 blood group transferase can be used as a marker independent of blood group molecules for confirming a suspected acquired B.


Degradation of oxytocin by the human placenta: effect of selective inhibitors.

The hydrolysis of oxytocin by human placental subcellular fractions was studied in the presence of selective inhibitors by measuring liberated amino acids by high performance liquid chromatography (HPLC). Oxytocin degradation by microsomal and lysosomal fractions was inhibited by bestatin, amastatin and puromycin. The IC50 values of these inhibitors on oxytocin degradation by both fractions were similar to those of these inhibitors on the human placental aminopeptidase M measured by L-Leu-p-nitroanilide as a substrate (LAP activity), which we reported previously. However, purified aminopeptidase M from human placental microsomal fractions could not liberate any amino acid from oxytocin. Since phosphoramidon (1 mumol/l), a putative metalloendopeptidase inhibitor, and N-benzylcarbonyl-valyl-prolinal (Z-Val-prolinal) (14 mumol/l), a selective inhibitor of post-proline endopeptidase, could not significantly influence the degradation of oxytocin by either subcellular fractions, neither enzyme seems to be actively involved in oxytocin degradation. These results strongly suggested the existence of oxytocinase(s) other than the above three enzymes in microsomal and/or lysosomal fractions of human placenta.


Actions of serotonin antagonists on cholera-toxin-induced intestinal fluid secretion.

The effects of several 5-hydroxytryptamine (5-HT) receptor antagonists were tested in rats in vivo on the intestinal fluid secretion evoked by cholera toxin. Five receptor antagonists were used, namely 2-bromolysergic acid diethylamine (2-bromo-LSD), granisetron, ketanserin, methysergide and ondansetron. The drugs were used in doses that inhibited the arterial hypertension and/or bradycardia evoked by 5-HT given i.v. Granisetron and ondansetron markedly diminished cholera-toxin-evoked secretion, whereas ketanserin was without any effect. Methysergide also diminished cholera-toxin-induced fluid secretion particularly when the drug was given as an i.v. infusion. The results are considered in relation to the pathophysiology of cholera secretion and to the current views of receptor subtypes for 5-HT. It is proposed that the receptor involved is a 5-HT3 receptor, possibly also a receptor of the 5-HT1 type. Results from experiments in which 5-HT (20 mM) was placed in the intestinal lumen to evoke an intestinal secretion suggest that the 5-HT3 receptor is located in the villus tissue. It was also demonstrated that zimeldine, an inhibitor of presynaptic 5-HT reuptake, diminished choleraic secretion, an effect that may be ascribed to a 5-HT tachyphylaxis caused by an accumulation of 5-HT in a synaptic cleft.


[Immunochemical detection of the proliferating cell nuclear antigen (PCNA/cyclin) in ocular tissues].

The authors developed radioimmunoassay (RIA) for proliferating cell nuclear antigen (PCNA/cyclin), which a protein synthesizes in phase with DNA, as a marker for cell replication. We used it to detect the amounts of PCNA and estimated rates of cell replication in ocular tissues (cornea, lens epithelium, retina-plus-choroid and optic nerve) and in non-ocular tissues (skin, skeletal muscle, brain, thymus, small intestine and liver) in rats. Antisera were raised in rabbits to oligopeptide fragment of human PCNA (hPCNA): the C-terminal fragment [Tyr254]-hPCNA (254-261). For RIA the synthetic peptides were used as standards and radioiodinated [125I-Tyr254]-hPCNA (254-261) as radioligands. The IC50 was 30-40 f mole/tube with a sensitivity 0.5-1.0 f mole/tube for this assay. Tissue extracts in 1 M acetic acid were digested with trypsin to release fragment (255-261) from whole PCNA and were assayed by this method. The highest amounts of immunoreactive PCNA were found in lens epithelium and skin, while the lowest amounts were found in optic nerve and muscle. The cornea showed a higher value than retina-plus-choroid or optic nerve. A high cell replication in the lens epithelium was revealed.


Effect of beta-adrenoceptor blockade on dipyridamole-induced myocardial asynergies in coronary artery disease.

Twenty-one patients with angiographic evidence of significant coronary artery disease, and positive dipyridamole echocardiographic test results at basal condition and after 7 days of placebo treatment were prospectively studied to see whether beta blockade modifies the effects of dipyridamole echocardiographic testing on regional myocardial contractility. Patients were randomized to propranolol (120 mg/day) or placebo treatment in 3 divided doses for 7 days, after which each patient crossed over to the alternate regimen. Dipyridamole-echocardiographic testing was repeated at the end of each treatment. Propranolol abolished new mechanical signs of transient dipyridamole-induced ischemia (new wall motion abnormalities or an increase in degree of basal asynergies, or both) in 13 of 21 patients. The remaining 8 patients had positive results on dipyridamole echocardiographic testing after the propranolol treatment period. At basal conditions both heart rate and rate-pressure product were significantly reduced with propranolol; there was also a significant decrease in these parameters at peak dipyridamole infusion. At peak dipyridamole infusion heart rate and rate-pressure product were significantly lower in patients with negative than in those with positive echocardiographic test results after propranolol. Our data show that administration of beta blockade significantly reduces the development of transient dipyridamole-induced myocardial asynergies, the earliest markers of acute myocardial ischemia, detected with 2-dimensional echocardiography.


Deletion of the Hunter gene and both DXS466 and DXS304 in a patient with mucopolysaccharidosis type II.

Hunter syndrome is an X-linked mucopolysaccharidosis due to deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). A cDNA clone containing the entire coding region of the human IDS gene, mapped in Xq28, has been used as molecular probe to study a patient with Hunter syndrome. A submicroscopic deletion has been detected that spans the IDS gene as well as DXS466 and DXS304, 2 loci mapped probably not more than 900 kb from the IDS locus. A detailed clinical description of the patient is provided and his phenotype is compared to that of other patients with IDS deletion described recently. By following the segregation of a restriction fragment length polymorphism at the IDS locus in the patient's family, our data suggest that the deletion occurred in the germ cells of the patient's grandfather.


First prenatal diagnosis of X-linked lymphoproliferative disease.

A family study was performed in order to diagnose X-linked lymphoproliferative (XLP) disease in a fetus. The molecular genetic analysis indicated that the fetus, as well as its healthy 7-year-old brother, inherited XLP. Analysis of immunoglobulin subclasses from the 7-year-old brother supported the DNA-based diagnosis. This is the first XLP family of African descent.


Dexamethasone prevents autoimmune nephritis and reduces renal expression of Ia but not costimulatory signals.

Although glucocorticoids are a conventional treatment for lupus nephritis, the cellular and molecular mechanisms responsible for preventing renal injury are unknown. MRL-lpr mice develop an aggressive autoimmune nephritis. As these mice become nephritic, there is an increase in the renal expression of molecules that permit or facilitate immune interactions, including MHC class II (Ia) antigens, intercellular adhesion molecule-1 (ICAM-1), and pro-inflammatory cytokines. Because dexamethasone (Dex) alters Ia antigen expression and suppresses cytokine generation, the authors prophylactically treated MRL-lpr mice and investigated the relative importance of these molecules in inducing renal injury. MRL-lpr mice given Dex (0.4 mg/kg/d) from age 6 weeks were killed 4, 8, and 16 weeks after the initiation of therapy, and tissue was removed for histology and extraction of total RNA. Dex prevented lymphadenopathy and renal injury. DEX eliminated the marked Thy 1.2+ lymphocytic infiltrates within the kidney and preserved normal renal histology and urinary protein levels. Northern blot analysis of steady-state mRNA transcripts indicated Dex suppressed a four-fold increase in kidney major histo-compatibility complex class II (Ia) molecule antigen mRNA seen by age 22 weeks (Ia/beta-actin ratios = 0.64 +/- 0.50 versus 2.32 +/- 0.48, P less than 0.01), but did not alter the costimulatory molecules ICAM-1 or tumor necrosis factor alpha (TNF alpha). Although all of these molecules are important mediators of inflammation, autoimmune nephritis was ameliorated without alteration of TNF alpha gene transcription or ICAM-1 transcription and surface expression. This study suggests that the benefit of steroids in nephritis stems from preventing lymphocyte infiltration into the kidney and decreasing immune interactions by limiting Ia expression.


An overdose of ecstasy. A role for dantrolene.

An overdose of the semisynthetic, hallucinogenic amphetamine 3,4-methylenedioxymethamphetamine resulted in convulsions, hyperthermia, hyperkalaemia and rhabdomyolysis. The patient's management, which included the use of dantrolene, is discussed.


Riboflavin 5'-pyrophosphate: a contaminant of commercial FAD, a coenzyme for FAD-dependent oxidases, and an inhibitor of FAD synthetase.

Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94% pure, the remaining 6% being composed of four or five minor contaminants which can be separated from FAD by reverse-phase high-performance liquid chromatography. FAD purified in this manner has been shown to be 100% pure. One of the contaminants has been identified as riboflavin 5'-pyrophosphate (RPP) by spectroscopic and chemical methods of analysis. This compound has been shown to exhibit biological activity as a weak cofactor for two FAD-requiring enzymes. With the apoprotein of porcine D-amino-acid oxidase, values determined for RPP were 8.4 microM for Km and 0.10 for Vmax compared to 0.47 microM and 0.28 (36 U/mg), respectively, for FAD. With fungal glucose apooxidase, values determined for RPP were 474 nM for Km and 0.02 for Vmax and 45 nM and 0.09 (105 U/mg), respectively, for FAD. RPP can also inhibit FAD biosynthesis. For bovine liver FAD synthetase, a Ki value for RPP against FMN was determined to be 9 microM where Km for FMN was 5.5 microM. These studies illustrate the value of riboflavin 5'-pyrophosphate as a flavin analog for use in the study of structure/function relationships within certain flavin-dependent enzymes.


Responses of plasma adrenocorticotropic hormone, cortisol, and cytokines during and after upper abdominal surgery.

There is currently accumulating evidence for bidirectional communication between the neuroendocrine and immune systems. Various cytokines have been suggested to be involved in the stimulation of stress hormone secretion during the times of infection and inflammation. To assess the possible involvement and pathophysiologic significance of cytokines in the mechanisms responsible for the perioperative stress response of the hypothalamo-pituitary-adrenal axis, we observed the changes of plasma adrenocorticotropic hormone and cortisol levels together with those of plasma endotoxin and cytokine levels. In patients undergoing pancreatoduodenectomy, perioperative stimulation of adrenocorticotropic hormone and cortisol secretion was accompanied by a significant elevation of plasma cytokine levels. Application of epidural block up to the upper thoracic levels failed to suppress this stress response effectively. In patients undergoing unilateral total hip replacement, the response of plasma hormone levels was smaller and briefer with no significant increase of plasma cytokine levels. Application of epidural block up to the lower thoracic levels suppressed this hormonal response almost completely. In patients undergoing pancreatoduodenectomy, a significant elevation of plasma endotoxin level was followed by a gradual but significant elevation of plasma tumor necrosis factor alpha and interleukin-6 levels. It seems likely that the stimulatory effects of these cytokines on the secretion of adrenocorticotropic hormone and cortisol might be involved in the development of the greater and more prolonged stress response of hypothalamo-pituitary-adrenal axis. Our present study suggests that not only neural input from the surgical wound but also stimulation of cytokine production were responsible for the development of the stress response of the hypothalamo-pituitary-adrenal axis during and after upper abdominal surgery.(ABSTRACT TRUNCATED AT 250 WORDS)


Role of guanylate cyclase-cGMP systems in halothane-induced vasodilation in canine cerebral arteries.

The cellular mechanisms through which halothane dilates blood vessels remain largely unknown. The present studies were designed to determine the effects of 0.59 and 0.9 mM halothane (equivalent to 2.0% and 3.0%, respectively) on tissue cyclic guanosine 3,5-monophosphate (cGMP) level and guanylate cyclase enzyme activity in canine middle cerebral arteries. Rings of cerebral arteries preconstricted with 5-hydroxytryptamine (0.2 microM) were exposed for 15 min to low or high concentrations of halothane or for 5 min to sodium nitroprusside (50 microM). The vessels were instantaneously frozen by immersing them in liquid N2; they then were homogenized, and the tissue cGMP levels were determined using radioimmunoassay. Halothane produced 2.23 +/- 0.44- and 4.47 +/- 0.87-fold increases in tissue cGMP levels over control at 0.59 and 0.9 mM, respectively. Sodium nitroprusside, a nitrovasodilator, also increased the tissue cGMP level 7.80 +/- 1.36-fold over the control value. To understand better the mechanisms of halothane-induced increase of tissue cGMP level, the effects of this anesthetic agent on guanylate cyclase enzyme activity were examined. Halothane, unlike sodium nitroprusside, did not modulate the activity of the soluble guanylate cyclase enzyme. However, halothane (1.0 mM), like atrial natriuretic factor (5 microM), stimulated the particulate guanylate cyclase enzyme activity. LY-83583 (6-anilino-5,8-quinolinedione, 10 microM), an agent that inhibits soluble guanylate cyclase activity, significantly reduced the response of the vessels to calcium ionophore (A23187, 0.4 microM), an endothelium-dependent vasodilator, without producing a significant effect on halothane-induced vasodilation. These results suggest that halothane-induced vasodilation of cerebral blood vessels is partly mediated by an increase in tissue cGMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Intrathecal clonidine and the response to hemorrhage.

Intraspinally administered alpha 2-adrenergic agonists are being examined for postoperative analgesia, yet their effects on the hemodynamic response to acute hemorrhage have not been examined. In this study chronically prepared conscious sheep received thoracic intrathecal saline or clonidine 300 micrograms followed in 15 min by rapid removal of 1,000 ml blood. In saline-treated ewes blood pressure was maintained and heart rate steadily increased during hemorrhage of up to 700 ml blood, with further blood removal resulting in rapid decreases in both variables. In contrast, heart rate never increased and blood pressure was maintained only up to 400 ml blood loss in animals receiving intrathecal clonidine. Compared to saline controls, clonidine did not alter blood pressure or heart rate at the end of hemorrhage or during blood pressure restitution during the next hour. Clonidine inhibited the increase in plasma epinephrine at the end of hemorrhage without altering plasma norepinephrine, vasopressin, renin, or atrial natriuretic factor. Intrathecal idazoxan, a specific alpha 2-adrenergic antagonist, reversed clonidine's effect on blood pressure during hemorrhage. Intravenous DG-5128, a poorly lipid-soluble alpha 2-adrenergic antagonist, also reversed clonidine's effect and additionally completely blocked any reduction in blood pressure and heart rate during hemorrhage. These data suggest that intrathecal clonidine interferes with maintenance of blood pressure during hemorrhage, likely because of a spinal sympatholytic effect, but does not affect the ultimate decrease in blood pressure after rapid removal of 1,000 ml blood. This difference in effect during the two phases of hemorrhage can be explained by the relative importance of the sympathetic nervous system in each.


Premedication with oral dexmedetomidine alters hemodynamic actions of intravenous anesthetic agents in chronically instrumented dogs.

Dexmedetomidine (the D-stereoisomer of medetomidine), a highly selective alpha 2-adrenoceptor agonist, has been demonstrated to produce analgesia and sedation and attenuate hemodynamic responses to emergence from inhalational anesthetics, which suggests a potential use for this drug as a premedicant for general anesthesia. The authors examined hemodynamic interactions between dexmedetomidine and three commonly used intravenous anesthetic agents with markedly different hemodynamic effects. Conscious, chronically instrumented dogs received intravenous induction doses of ketamine, propofol, or etomidate, followed by continuous infusions of each drug at four different doses for 15-min intervals on different days. Studies in six separate groups (range, 9-12 dogs/group) with and without pretreatment with oral dexmedetomidine (20 micrograms/kg) were completed. Heart rate, arterial pressure, left ventricular pressure, rate of increase of left ventricular pressure at 50 mmHg (dP/dt50), and cardiac output were continuously recorded. Dexmedetomidine administration caused a significant (P less than 0.05) decrease in heart rate, rate-pressure product, left ventricular dP/dt50, and cardiac output. Dexmedetomidine abolished or attenuated the increase in heart rate, rate-pressure product, cardiac output, and arterial pressure produced during induction of anesthesia with ketamine. After the dexmedetomidine pretreatment, continuous infusion of ketamine caused no increase in heart rate or rate-pressure product. However, ketamine significantly reduced left ventricular dP/dt50 compared to control in dogs premedicated with dexmedetomidine. Except for a significant reduction in systemic vascular resistance, dexmedetomidine did not significantly affect the hemodynamic response to induction of anesthesia with propofol. Similarly, dexmedetomidine did little to alter the hemodynamic response to induction of anesthesia with etomidate.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of 22 months of treatment with inhaled corticosteroids and/or beta-2-agonists on lung function, airway responsiveness, and symptoms in children with asthma. The Dutch Chronic Non-specific Lung Disease Study Group.

In a randomized double-blind multicenter clinical study, 116 children with asthma were randomly assigned to treatment with an inhaled beta-2-agonist (salbutamol 0.2 mg) plus an inhaled corticosteroid (budesonide 0.2 mg) three times a day (BA+CS) or to an inhaled beta-2-agonist (salbutamol 0.2 mg) plus a placebo three times a day (BA+PL). After a median follow-up time of 22 months, 26 patients receiving BA+PL (45%) had withdrawn from randomized treatment, mainly because of asthma symptoms, compared with three withdrawals in the patients receiving BA+CS (p less than 0.0001). The FEV1, expressed as a percentage of the predicted value for age, sex, and height, showed an absolute increase of 7.0% after 2 months of BA+CS compared with a decrease of 4.0% after 2 months of BA+PL. This 11% difference in percent predicted FEV1 (95% confidence interval, 7 to 15%; p less than 0.0001) was then maintained after a median follow-up period of 22 months. Postbronchodilator FEV1 showed an absolute increase of 3.7% predicted within 2 months in patients receiving BA+CS and an absolute decrease of 1.1% predicted in children receiving BA+PL (p = 0.0005). Thereafter, this difference between the two treatment groups was maintained. Average peak expiratory flow rate (PEFR) increased from baseline by 36.6 L/min in the BA+CS group compared with 3.7 L/min in the BA+PL group (p = 0.003). This difference then remained for the median follow-up time of 22 months.(ABSTRACT TRUNCATED AT 250 WORDS)


Canine in vivo tracheal chemotaxis of eosinophils to antigen in sensitized dogs: inhibition by a steroid, a systemic lazaroid U-78517F, and several topical H1 antihistamines.

Inflammation of the airways contributes to the multicomponent disease known as asthma. The primary cells that infiltrate the airway in response to antigen exposure are neutrophils and eosinophils. Eosinophils have been implicated in much of the histopathology of the airway following infiltration and degranulation. We used topical antigen exposure in the trachea of sensitized beagle dogs to study the kinetics of eosinophil infiltration with a modified double balloon endotracheal tube technique. After establishment of the eosinophilia, we used inhibitors with known actions to implicate certain mediators in the cellular response. Beagle dogs were sensitized to ascaria ova by feeding them orally by gavage. After 6 wk, challenge with 2.0 micrograms/ml ascaris antigen protein via the tracheal chamber resulted in a rapid (maximal in 4 h) and repeatable influx (p less than 0.05 versus vehicle) of eosinophils that was faster and larger than that of exogenously added LTB4 or PAF exposure. After 4 to 6 separate (every 2 wk) antigen challenges in which the response varied by (SEM +/- 13 to 50%), the individual dogs were rechallenged in the presence of various inhibitors administered either topically in the perfusate or systemically. The inhibitors that were effective in blocking the eosinophil influx by a statistically significant amount were: (1) the lazaroid U-78517F orally 0.5 to 30 mg/kg-18 h (2) Medrol acetate 5 mg/kg intramuscularly-18 h, (3) H1-antihistamines topically (10(-6) M) Astemizole, Cetirizine, and Mepyramine, also orally 30 mg/kg, and Azelastine. Inhibitors falling to inhibit influx topically were a LTB4 receptor antagonist (U-75302) and U-80271 (Merck L-652731), a PAF antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)


[Chronic myelogenous leukemia with blastic crisis in which expression of P-glycoprotein was associated with resistance to chemotherapy].

A 55-year-old woman with chronic myelogenous leukemia developed a lymphoid blast crisis (BC) 10 months after diagnosis. By using immunoblotting with a monoclonal antibody against P-glycoprotein (P-gp) C219, her leukemia cells from the first and 3rd crises were shown to be negative for the P-gp, while the cells of the 4th crisis were detected to have a high level of P-gp. This patient did not respond to chemotherapy with several anti-cancer agents in the 4th crisis, although complete remission was achieved in the first, second and third crises after administration of agents including vincristine and prednisolone. Therefore the expression of P-gp in the 4th BC might have been closely related to the resistance to chemotherapy.


Cryptorchidism: a prospective study of 7500 consecutive male births, 1984-8. John Radcliffe Hospital Cryptorchidism Study Group.

A total of 7441 boys were examined for cryptorchidism at birth and, if present, again at 3 months of age. After excluding boys with severe congenital malformations noted at birth, the cryptorchidism rates at 3 months in babies weighing less than 2000 g, 2000-2499 g, and greater than or equal to 2500 g were 7.7%, 2.5%, and 1.41% respectively. The overall rate was 1.55%. The cryptorchidism rate at birth had increased by 35.1% and at 3 months by 92.7%, over Scorer's rates in the 1950s. Part of these increases may be attributable to differences in neonatal mortality, but the increases in babies weighing 2500 g or more of 50.2% at birth and 77.4% at 3 months are unlikely to be overestimates. At birth 1.92% of boys had bilateral cryptorchidism and 3.0% unilateral cryptorchidism. Boys with cryptorchidism at 3 months were more likely to have hypospadias, a small scrotum, and poor scrotal rugation compared with boys having normally descended testes at birth. Factors predicting descent by 3 months in babies cryptorchid at birth are birth weight, laterality and scrotal size, babies with low birth weight, bilateral cryptorchidism, and normal scrotal size being more likely to have normally descended testes by 3 months. Descent by 3 months was more likely the lower the testis along the normal pathway of descent. The orchidopexy rate at an average age of 3 years was 1.24%. This is substantially lower than in other series and lower than our estimated rate of 2.9% using Hospital In-Patient Enquiry data for England and Wales.


Prevention of exercise induced asthma by inhaled salmeterol xinafoate.

The effect of inhaled salmeterol xinafoate, a long acting beta 2 agonist, on exercise induced asthma was studied in a double blind, crossover, and placebo controlled trial. Thirteen asthmatic children with a fall of at least 15% in their forced expiratory volume in one second (FEV1) after a standard exercise test on a motorised treadmill, on separate days performed the same test 1, 5, and 9 hours after a single dose of 50 micrograms salmeterol or placebo. FEV1 was measured before treatment, and before and for 30 minutes after each exercise test. After placebo the number of children with exercise induced asthma was: 10 at 1 hour, 11 at 5 hours, and 12 at 9 hours. Salmeterol prevented exercise induced asthma in all 13 children studied, at 1, 5, and 9 hours. Mean maximum falls in FEV1 after exercise were at 1 hour: salmeterol 2.7% and placebo 24.6%, 5 hours: salmeterol 5.3% and placebo 22.7%; and 9 hours: salmeterol 3.4% and placebo 26.6%. After salmeterol the mean increase in FEV1 was 17.8% at 1 hour, 19.6% at 5 hours, and 19.2% at 9 hours. Inhaled salmeterol prevents exercise induced asthma and produces significant bronchodilatation for at least 9 hours.


Blood phobics and nonphobics: psychological differences and affect during exposure.

This study compared psychological dimensions of blood phobics and nonphobic controls, examined affect in response to phobic and neutral stimuli, and investigated the relationship between reported feelings of faintness and blood pressure. Blood phobics (24 adults with extreme Mutilation Questionnaire scores) and 24 nonphobics completed several psychological measures and viewed one of two 60 sec surgery scenes and a 60 sec neutral scene in counterbalanced order. Subjective, psychophysiologic, and motoric measures of affect were assessed. On questionnaires, phobics reported greater anxiety sensitivity, empathic distress, fear and insecurity, and nightmares, but no difference in autonomic arousal, muscle tension, motion sickness, or other empathy domains. During surgery scenes, phobics had more negative affect than controls; however, phobics were more anxious during only one of the two surgeries, and often only when the surgery was presented prior to the neutral scene. Fainting did not occur, and self-reported feelings of faintness were unrelated to blood pressure changes. The findings highlight the lack of information on blood phobic stimulus properties, fainting's relationship to self-reports and blood pressure, and the specific emotion experienced in blood phobia.


Restriction fragment analysis of the secalin loci of rye.

Analyses of wheat/rye addition lines by Southern blotting confirmed the presence of sequences related to the Sec 1, Sec 2, and Sec 3 loci on chromosomes 1R and 2R. Comparison of the 1R and 2R addition lines allowed the identification of gamma-secalin genes at Sec 1 and Sec 2, respectively, while omega-secalin and gamma-secalin genes at Sec 1 were discriminated by comparative hybridization with three probes: omega-secalin, total gamma-secalin, and 3' gamma-secalin. The high molecular weight (HMW) secalin genes at Sec 3 were identified using a homologous HMW subunit probe from wheat. Gene copy numbers were estimated as about 40-60 for omega-scalins, 5-10 for gamma-secalins, and 2 for HMW secalins. Comparison of individual plants of cv. Gazelle showed a high degree of polymorphism, particularly for sequences related to omega-secalins and HMW secalins.


Rationally designed selective inhibitors of trypanothione reductase. Phenothiazines and related tricyclics as lead structures.

Trypanothione reductase, an essential component of the anti-oxidant defences of parasitic trypanosomes and Leishmania, differs markedly from the equivalent host enzyme, glutathione reductase, in the binding site for the disulphide substrate. Molecular modelling of this region suggested that certain tricyclic compounds might bind selectively to trypanothione reductase without inhibiting host glutathione reductase. This was confirmed by testing 30 phenothiazine and tricyclic antidepressants, of which clomipramine was found to be the most potent, with a K(i) of 6 microM, competitive with respect to trypanothione. Many of these compounds have been noted previously to have anti-trypanosomal and anti-leishmanial activity and thus they can serve as lead structures for rational drug design.


Cell cycle-dependent changes in tissue transglutaminase mRNA levels in bovine endothelial cells.

The pattern of transglutaminase gene expression through the cell cycle was examined by Northern blot analysis using cultured bovine endothelial cells and a cDNA probe. When the cells reached confluency or were arrested in G0/G1 phase by nutrition deprivation, transglutaminase mRNA rose to a very high level; S- and M-phase extracts showed high and low levels, respectively. Subcellular localization studies by sucrose gradient centrifugation and immunostaining demonstrated that the majority of transglutaminase is present in cytosols throughout the cycle. The cell cycle-dependent changes in the transglutaminase mRNA levels strongly support the implicated involvement of the enzyme in cell growth, differentiation, and senescence.


The N-terminal hexapeptide fragment of IGF II stimulates thymidine incorporation into fibroblasts.

We synthesized the N-terminal hexapeptide fragment of IGF II to study potential binding to NMDA receptors in analogy to the N-terminal tripeptide of IGF I. The amino acid sequence of the hexapeptide is furthermore identical with the C-terminal sequence of the casiragua insulin B chain. The hexapeptide did not bind to the NMDA receptors, but was found to promote [3H]-thymidine incorporation into fibroblasts at concentrations of 10(-8) - 10(-5) M in a dose-dependent manner. Since [125I]-hexapeptide did not bind to IGF receptors, indirect competition studies using either labelled IGFs or insulin had to be used. The competition of hexapeptide at a concentration of 10(-5) M with labelled IGF I or II was about equal to that of 10(-9) M IGF I or II. IGF receptors were apparently up-regulated by the hexapeptide, as has also been described for insulin. When using casiragua insulin as labelled ligand, IGF II and casiragua insulin competed with equal potency, whereas the hexapeptide at 10(-7) M caused an apparent up-regulation of the casiragua insulin binding sites. Our results that the hexapeptide stimulates [3H]-thymidine incorporation and up-regulates IGF II and casiragua insulin binding sites may be connected to one or several of the following findings: the hystricomorph insulins--of which the casiragua insulin is a member--stimulate DNA synthesis to a greater extent than other insulins; the insulin and type 1 IGF receptor binding regions are localized predominantly in the C-terminal region of the insulin B chain; and the "cooperative" site regulating the affinity of the insulin receptor is also located in the C-terminal region of the insulin B chain. Further experiments will be needed to clarify the exact mechanism.


Effect of dynorphin A(1-13) on cardiomyocytes in culture: modulation of the response to increased extracellular calcium, but no effect on intrinsic cardiac contractile frequency or the response to isoproterenol or increased extracellular potassium.

The purpose of this study was to determine whether the endogenous opioid peptide dynorphin A(1-13) has a direct effect on the heart or acts to modulate the cardiac chronotropic response to calcium, potassium, or beta-adrenergic receptor stimulation. Spontaneously contracting myocardial cell aggregates were prepared from 7-day-old chick embryos and were maintained in culture for 72 h before study. Dynorphin A(1-13), 10(-8) to 10(-6)M, did not alter spontaneous contractile frequency. Increases in [Ca2+]o spontaneously suppressed cardiac contractile frequency, and dynorphin A(1-13) significantly (p less than 0.05) enhanced this response. Nifedipine, 10(-8) M, antagonized the effect of increased [Ca2+]o on cardiac contractile frequency, but did not block the action of dynorphin A(1-13) to accentuate the effect of increasing [Ca2+]o. Dynorphin A(1-13) did not alter the significant (p less than 0.05) increase in contractile frequency produced by beta-adrenergic receptor stimulation by isoproterenol, or the suppression in contractile frequency produced by increases in extracellular potassium ([K+]o). These data indicate that dynorphin A(1-13) does not act directly on the cardiac myocyte to alter cardiac contractile frequency or alter the response to increases in [K+]o or to isoproterenol, but that dynorphin A(1-13) does modulate the response to increases in extracellular calcium.


Evidence by in situ hybridization that c-erbB-2 proto-oncogene expression is a marker of malignancy and is expressed in lung adenocarcinomas.

In order to identify potential markers of malignancy in diagnostic respiratory cytopathology, c-myc and c-erbB-2 proto-oncogene expression was studied in fine needle aspirates from 14 consecutive fresh operation tissue samples (after surgical removal) representing lung tumors and a variety of other cell samples by in situ hybridization of 35S-labeled antisense and sense RNA c-myc and c-erbB-2 specific proto-oncogene probes. All 14 lung tumors showed c-myc expression and eight also showed c-erbB-2 expression. On average, the c-myc expression was about 4 times higher than that of c-erbB-2 (P less than 0.001). c-erbB-2 expression, confirmed also as a cytoplasmic membrane-bound reactivity by immunohistochemical stainings for c-erbB-2 oncoprotein, was significantly related to adenocarcinoma (P less than 0.025), whereas increasing tumor size correlated significantly with increasing c-myc expression (P less than 0.05). On average, all the tumor cell lines showed 2-fold expression of c-myc compared with the lung tumors (P less than 0.025). c-erbB-2 expression was found in six of 11 cell lines. High c-myc proto-oncogene expression was also found in broncho-epithelial cells and alveolar macrophages, and a low expression was found in lymphocytes but not in neutrophils, while none of these cells showed c-erbB-2 proto-oncogene expression. Our results demonstrate extensive c-myc proto-oncogene expression in both malignant and non-neoplastic proliferating cells, but not in terminally differentiated cells such as neutrophils. Therefore c-myc expression must also be related to general cell proliferation and not only malignancy per se. In marked contrast, c-erbB-2 proto-oncogene expression was found only in adenocarcinoma cells, and thus can be used as a marker for malignancy in diagnostic respiratory cytopathology.


Carbohydrate antigens of embryonal carcinoma cells: changes upon differentiation.

Embryonal carcinoma (EC) cells, the malignant stem cells of teratocarcinomas, resemble early embryonic cells morphologically, developmentally, and with respect to several cell surface characteristics. EC cells often differentiate into a variety of cell types when treated with chemical agents such as retinoic acid, or when placed in a normal embryonic environment. Developmentally regulated surface antigens of EC cells and their differentiated derivatives have been defined using monoclonal antibodies. Many of these "differentiation antigens" have proved to be oligosaccharide chains carried on membrane glycolipids and/or glycoproteins. Our analyses of glycolipid composition in a pluripotent human EC cell line, NTERA-2, have revealed a complex set of glycoslylation changes that occur during cellular differentiation. Like early embryos, undifferentiated NTERA-2 EC cells express predominantly globo-series glycolipids. However, once differentiation is initiated by addition of retinoic acid there is a marked shift of cellular glycolipids from globo-series to lacto- and ganglio-series. Distinct subsets of differentiated cells are characterized by their expression of different patterns of glycolipid antigens. These changes in cell surface phenotype may serve to mark cellular identity and facilitate morphogenetic cell interactions during embryonic development.


Microbial interaction with animal cell surface carbohydrates.

Microbes have selected primarily carbohydrates for attachment to host animal cells. Recent studies have revealed essential characteristics in the recognition of receptor carbohydrates. Of importance is the property of recognizing also sequences placed inside an oligosaccharide chain, which differs from most animal antibodies. This is the basis for series of isoreceptors with the minimum receptor sequence in common but with separate neighbouring groups. There are families of microbial ligands that show different preferences for members within one series of isoreceptors, indicating only slight differences in the complementary binding sites of the proteins. Such differences may explain shifts in the selectivity of separate host tissues for infection. A second characteristic is the low affinity interaction often found where simple receptor-containing saccharides are unable to inhibit attachment. Technical possibilities are rapidly developing for the design of synthetic receptor analogues to be used in the therapy of clinical infections. This is urgently needed in cases where no rational therapy exists today.


Prevention of HIV-2 and SIV infections in cynomolgus macaques by prophylactic treatment with 3'-fluorothymidine.

The aim of this study was to determine the usefulness of human immunodeficiency virus type 2 (HIV-2) for in vivo evaluation of antiviral drugs in monkeys and to study if prophylactic treatment with 3'-fluorothymidine (FLT) could prevent infection against a low challenge dose of HIV-2 or simian immunodeficiency virus (SIV). Protection against infection was assessed by virus isolation and polymerase chain reaction (PCR) on monkey peripheral blood mononuclear cells (PBMC) as well as by antibody and viral antigen assays. Prophylactic treatment with FLT 3 x 5 mg/kg/day, starting 8 h prior to virus inoculation, prevented HIV-2 infection in 3 of 8 monkeys. In another experiment 2 of 4 monkeys resisted 2-10 monkey infectious doses (MID50) of SIV with the same prophylactic treatment. All control animals (HIV-2 n = 8, SIV n = 4) became infected. Thus, FLT treatment prevented HIV-2 and SIV infection in 5 of 12 animals.


Comparative accuracy of fixed partial dentures made as one-piece castings or joined by solder.

Four-unit fixed partial dentures were fabricated as a one-piece casting or as two-piece castings joined by soldering prior to the addition of porcelain. The accuracy of the two techniques was compared and evaluated using an anatomic cast-metal master model. All procedures, including SEM measurements, were performed directly on the master model. The cast one-piece units had smaller vertical marginal openings than did the soldered units. The rigid nature of the abutments evokes the problems occurring with osseointegrated implants.


Dopamine fiber growth induction by implantation of synthetic dopamine-containing microspheres in rats with experimental hemi-parkinsonism.

Injectable local drug delivery formulations-so-called microspheres have recently been developed, in which drugs are microencapsulated within biocompatible and biodegradable copolymer excipients like poly[DL-lactide-co-glycolide]. In view of its potential therapeutical usefulness, we have studied the microsphere methodology as a means to substitute for experimentally induced subnormal levels of endogenous dopamine (DA). Administration of 6-hydroxydopamine (6-OH-DA) unilaterally in the medial forebrain bundle of rats results in an up-regulation of postsynaptic receptors in the denervated striatum, functionally manifested as contralateral rotational behavior after apomorphine. DA microspheres were implanted in the denervated striatum. The majority of the rats displayed an attenuation of the contralateral rotational behavior induced by apomorphine up to 8 wk postimplantation. Immunocytochemical observations unexpectedly demonstrated growth of DA and tyrosine hydroxylase immunoreactive fibers in the denervated striatum. Interestingly, there was an apparent correlation between functional recovery and the degree of growth of DA fibers. The present results suggest that implantation of DA microspheres may promote DA fiber growth and extended recovery of surviving DA neurons, and, therefore, could be of therapeutic usefulness in Parkinson's disease.


Human pancreatic cancer cell lines do not express receptors for somatostatin.

The in vivo administration of somatostatin (SS) or its analogues is capable of suppressing the growth of pancreatic cancer in experimental animals. We examined the effects of SS-14 and its analogue RC-160 on the in vitro growth of two human pancreatic cancer cell lines MiaPaCa-2 and Panc-1 stimulated with epidermal growth factor (EGF) or insulin-like growth factor 1 (IGF-1). Neither SS-14 nor RC-160 inhibited the growth of either cell line. In contrast RC-160 did inhibit the EGF-stimulated growth of a rat pancreatic cancer cell line AR42J. Binding studies with 125I-Tyr11 somatostatin revealed the presence of a single class of high affinity binding sites with a Kd of 0.20 +/- 0.05 nM and a Bmax of 2.1 +/- 0.26 pmoles mg-1 protein on AR42J but not displaceable binding was observed on MiaPaCa-2 or Panc-1. We conclude that lack of receptors accounts for the failure of SS-14 and RC-160 to influence the growth of human pancreatic cancer in vitro. These results, taken together with other findings, lead us to question the therapeutic efficacy of somatostatin and its analogues as mono-therapy in the treatment of human pancreatic cancer.


Mdr1/P-glycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias.

In a variety of adult and childhood leukaemia cell samples collected at different states of the disease, we analysed in a series of sequentially performed slot-blot or Northern-blot hybridisation experiments the expression of genes possibly involved in multiple drug resistance (MDR) (mdr1/P-glycoprotein, DNA topoisomerase II, glutathione-S-transferase pi), and the expression of the DNA topoisomerase I and histone 3.1 genes. Occasionally, P-glycoprotein gene expression was additionally examined by indirect immunocytofluorescence using the monoclonal antibody C219. No significant difference in mdr1/P-glycoprotein mRNA levels between primary and relapsed state acute lymphocytic leukaemias (ALL) was seen on average. Second or third relapses, however, showed a distinct tendency to an elevated expression of this multidrug transporter gene (up to 10-fold) in part well beyond the value seen in the moderately cross-resistant T-lymphoblastoid CCRF-CEM subline CCRF VCR 100. Increased mdr1/P-glycoprotein mRNA levels were also found in relapsed state acute myelogenous leukaemias (AML), and in chronic lymphocytic leukaemias (CLL) treated with chlorambucil and/or prednisone for several years. Topoisomerase I and topoisomerase II mRNA levels were found to be very variable. Whereas in all but one case of CLL topoisomerase II mRNA was not detected by slot-blot hybridizations, strong topoisomerase I and topoisomerase II gene expression levels, frequently exceeding the levels monitored in the CCRF-CEM cell line, were seen in many cell samples of acute leukaemia. If topoisomerase II mRNA was undetectable, expression of topoisomerase I was clearly visible throughout. These observations might be valuable considering the possible treatment with specific topoisomerase I or topoisomerase II inhibitors. Significant positive correlations were found (i) for topoisomerase I and histone 3.1 gene expression levels in general (P less than 0.001), and (ii) in the CLL samples additionally for the expression levels of the mdr1 gene, and the histone 3.1, topoisomerase I, and glutathione-S-transferase pi genes, respectively.


P-glycoprotein expression in locally advanced breast cancer treated by neoadjuvant chemotherapy.

Using immunohistochemistry and the monoclonal antibody C219 we have investigated P-glycoprotein expression in 26 locally advanced breast cancers. Twenty four patients had received four cycles of chemotherapy (mitozantrone, mitomycin-C and methotrexate) prior to mastectomy; two received tamoxifen. Twelve tumours exhibited an objective response to the chemotherapy. A background pattern of isolated weakly positive (1+) stromal staining (myofibroblast) was observed in seven tumours, two of which had been treated by tamoxifen alone. Two of the tumours treated by induction chemotherapy showed positive staining (1+) within a very small number of isolated tumour cells (maximum of three) and macrophages. The significance of this staining is not clear although C219 may simply be cross reacting with myosin. We have failed to demonstrate a clear clinical utility for C219 in breast cancer, particularly regarding the identification of patients in whom MDR chemotherapy be avoided once metastases develop.


p53 protein expression in human breast carcinoma: relationship to expression of epidermal growth factor receptor, c-erbB-2 protein overexpression, and oestrogen receptor.

The expression of p53 protein, oestrogen receptor protein, epidermal growth factor receptor (EGFR) and overexpression of the c-erbB-2 oncoprotein was examined in a series of 149 primary symptomatic breast carcinomas. Expression of p53 was present in 62 of 146 cases (42.5%) of the invasive carcinoma and one of three cases (33.3%) of ductal carcinoma in situ (DCIS) examined. Statistical associations of tumour oestrogen receptor positivity and lack of p53 protein expression, chi 2 = 19.78 (d.f. = 1), P less than 0.001, positive tumour p53 status and poor tumour grade; chi 2 = 14.1 (d.f. = 2), P less than 0.001, EGFR expression chi 2 = 7.07, (d.f. = 1), P less than 0.01 and tumour c-erbB-2 protein overexpression; chi 2 = 4.61 (d.f. = 1), P = 0.032 were identified. Expression of p53 is rare in invasive lobular carcinoma of classical type (8.3% of cases examined) in contrast to other common types of mammary carcinoma. Non-significant trends of p53 protein expression and increased regional tumour recurrence; chi 2 = 3.20 (d.f. = 1), P = 0.074 and also poorer patient survival; chi 2 = 3.76 (d.f. = 1), P = 0.053 were identified. p53 protein expression is a common event in human breast cancer and is present in both DCIS and invasive mammary carcinoma. Abnormal expression of p53 protein is a feature of both in situ and invasive breast carcinoma, implying that the abnormal p53 protein expression may be implicated in the early stages of mammary carcinoma progression.


Glutathione S-transferase expression in the human testis and testicular germ cell neoplasia.

Glutathione S-transferase (GST) isoenzyme expression is altered in a variety of neoplasms and the enzymes are implicated in metabolism of carcinogens and resistance to drugs, including cisplatin. We have studied GST Alpha, Pi, Mu and microsomal isoenzyme expression by immunohistochemistry in normal and cryptorchid testes, intratubal germ cell neoplasia (ITGCN), seminoma and non-seminomatous germ cell tumours. In 16 stage II-IV malignant teratoma intermediate (MTI) both orchidectomy and post-treatment residual surgical masses were studied. All four isoenzymes were strongly expressed in Leydig and Sertoli cells. GST Pi was absent from normal spermatogonia but strongly expressed by the neoplastic germ cells of ITGCN and seminoma. GST Pi was strongly expressed in all elements of teratoma, irrespective of differentiation. There were no qualitative differences in expression between primary and post-chemotherapy metastases. GST Alpha expression in teratoma correlated with epithelial differentiation. GSTs may be important in normal spermatogenesis and protection of germ cells from teratogens and carcinogens. They may have a role in testicular tumour drug resistance but this role is not well defined. GST Pi is a new marker for ITGCN.


Comparison of ketanserin and celiprolol on regression of left ventricular hypertrophy in older hypertensive patients.

The effects of ketanserin, a specific serotonin2-receptor agonist, and celiprolol, a new, highly cardioselective beta 1 blocker with partial beta 2 agonist activity and peripheral vasodilating properties, on left ventricular (LV) structure and function were assessed in 60 older hypertensive patients (greater than 55 years) with clinical LV hypertrophy (LV mass index greater than 130 g/m2). The patients were studied using echocardiography after 1 month of placebo treatment, and 6 and 18 months of monotherapy with active drug. Ketanserin and celiprolol lowered blood pressure to normal levels. Heart rate did not change with ketanserin and fell moderately (-5%) with celiprolol (p less than .001). Regression of LV hypertrophy was achieved with the use of either medication (p less than .0001), although the magnitude of reduction in LV mass was greater with celiprolol at both 6 months (-10% vs -5%, p = .001) and 18 months (-13% vs -7%, p = .002). While LV volume did not change with either drug, celiprolol tended to decrease it, resulting in a 5% reduction in cardiac index (p = .01), which was associated with mild bradycardia. Ketanserin did not change LV ejection fraction, whereas celiprolol caused a slight (1.5%) long-term improvement (p = .003). Systolic wall stress and total peripheral resistance decreased with both agents (p less than .01), with no between-group differences. In conclusion, anti-hypertensive treatment of older persons with ketanserin or celiprolol achieves regression of LV hypertrophy without associated deleterious effects on LV function.


ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.

In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.


Transport properties of P-glycoprotein in plasma membrane vesicles from multidrug-resistant Chinese hamster ovary cells.

Multidrug resistant (MDR) cells overexpress a 170-180 kDa membrane glycoprotein, the P-glycoprotein, which is believed to export drugs in an ATP-dependent manner. Plasma membrane vesicles from the MDR CHRC5 cell line, but not the AuxB1 drug-sensitive parent, showed uptake of [3H]colchicine and [3H]vinblastine that was stimulated by the presence of ATP and an ATP-regenerating system. Steady-state uptake of drugs was achieved by 10 min and was stable for greater than 30 min. Non-hydrolysable ATP analogues were unable to support drug uptake, indicating that ATP hydrolysis is essential for transport. ATP-stimulated drug uptake appeared to result from drug transport into inside-out vesicles, since uptake was osmotically sensitive and could be prevented by detergent permeabilization. Steady-state uptake was half-maximal at 100 microM colchicine and 200 nM vinblastine and was inhibited by a 10-100-fold excess of MDR drugs and chemosensitizers, in the order vinblastine greater than verapamil greater than daunomycin greater than colchicine. In addition to being vanadate-sensitive, drug uptake was inhibited by 10-200 microM concentrations of several sulfhydryl-modifying reagents, suggesting that cysteine residues play an important role in drug transport. Vesicular colchicine was rapidly exchanged by an excess of unlabelled drug, demonstrating that drug association is the net result of opposing colchicine fluxes across the membrane.


Cell-surface changes induced by ectopic expression of the murine homeobox gene Hox-3.3.

Murine homeobox-containing genes (Hox genes) are postulated as playing key roles in the establishment of the anterior-posterior embryonic body axis, possibly providing cells with positional cues. Little is known, however, concerning how cells might respond to homeobox gene expression to interpret these cues. Since changes in the cell-surface are central to many processes in early development we reasoned that cells expressing different complements of Hox genes might have different surface properties. In order to investigate this we have used the sensitive, non-disruptive technique of multiple two-phase aqueous partition, which is able to detect small differences on the surface of intact cells. Using this technique we have found that ectopic expression of the murine Hox-3.3 gene in cultured cells induces reproducible changes in the cell surface. Changes only occurred above a threshold level of gene expression, but above this level a correlation between surface change and gene expression was seen. The implications for the establishment of a 'Hox' code of homeobox genes acting to specifically change cell-surface properties are discussed.


Neurotransmitter antagonists block some odor responses in olfactory receptor neurons.

The first step in olfactory transduction is the recognition of odor molecules by membrane bound receptors belonging to the superfamily of G-protein coupled receptors; other members of this family are involved in neurotransmission. Based on the considerable homology between individual members of this family, we have investigated the ability of well-known neurotransmitter antagonists to block the olfactory response. Adrenergic and muscarinic antagonists were found to block some odor induced currents (45-55%) with an IC50 between 15 and 75 microM. By contrast, antagonists of glutamate and GABA receptors, which do not belong to this receptor superfamily, were ineffective. These results suggest that further pharmacological analysis may be useful for characterizing and classifying the family of odor receptors.


Kainate/glutamate-induced changes in intracellular calcium and pH in leech glial cells.

Kainate evokes a non-desensitizing membrane depolarization in neuropile glial cells of the leech Hirudo medicinalis. We measured membrane potential and intracellular pH, pH(i), using double-barrelled pH-sensitive microelectrodes, and intracellular calcium, Ca2+i, using the change in fluorescence ratio of intracellularly injected fura-2, in these glial cells in situ. 20-50 microM kainate produced a depolarization of 18-28 mV and a decrease of pH(i) by 0.27 +/- 0.07 pH units. Ca2+i increased by 306 +/- 128 nM upon kainate, which could be inhibited by the non-NMDA antagonist CNQX. Glutamate (0.1 mM) also produced a fall in pH(i) and a rise in Ca2+i, which were however, much smaller. Quisqualate and N-methyl-D-aspartate had only small or no effects on membrane potential, pH(i) or Ca2+i. It is concluded that leech neuropile glial cells have a kainate-type glutamate receptor, which mediate significant transients of intracellular H+ and Ca2+.


Granulocyte-macrophage colony-stimulating factor induces neutrophil adhesion to pulmonary vascular endothelium in vivo: role of beta 2 integrins.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) causes upregulation of neutrophil surface CD11b/CD18 expression, and enhances the adhesion of neutrophils to cultured human endothelial cells in vitro. Systemic administration of GM-CSF results in a rapid, transient decrease in circulating phagocyte numbers. Using a nonhuman primate model (Cynomolgus), we provide histologic evidence that this transient leukopenia is associated with the margination of neutrophils in the pulmonary microcirculation. In four animals receiving 2 to 15 micrograms/kg recombinant human GM-CSF (rhGM-CSF), light microscopic sections of lung contained 36 +/- 8, 17 +/- 7, 21 +/- 6, and 15 +/- 8 (mean +/- SD, n = 20) neutrophils within a graticule grid, as compared with two control animals receiving saline injections whose lung sections contained 2.1 +/- 1.6 and 3.1 +/- 2.1 (mean +/- SD, n = 20) neutrophils within the same grid. Scanning electron microscopy shows activated leukocytes adherent to pulmonary vascular endothelium, but no morphologic evidence of endothelial damage, and no migration of cells into the extravascular space. Margination is associated with an increase in surface expression of CD11b/CD18 on circulating phagocytes, which could contribute to the adhesion to capillary endothelial cells, but CD11b/CD18 levels remain elevated even when demargination is complete. In vitro, monoclonal antibodies (MoAbs) to CD18 and CD11b were able to inhibit neutrophil aggregation and adhesion to endothelium. FMLP-induced neutrophil aggregation was inhibited by 39.8% +/- 11.5% and 44.8% +/- 12.3%, respectively, by MoAbs to CD18 and CD11b (P less than .0005, n = 4 for both); a similar effect was demonstrated on TPA-induced aggregation. MoAb CD18 reduced the adhesion of unstimulated neutrophils to endothelium by 44% (P less than .01, n = 7), and inhibited the amount of GM-CSF-stimulated adhesion by 74% (P less than .001, n = 7), while MoAb to CD11b produced a reduction of unstimulated neutrophil adhesion by 30%, and of GM-CSF-stimulated adhesion by 40% (P less than .01, n = 5, for both). However, when administered in vivo, MoAb CD18 produced only a small, albeit significant, amelioration of GM-CSF-induced margination in vivo, while MoAb CD11b was without effect. These results show that GM-CSF-induced transient leukopenia is associated with enhanced neutrophil adherence to pulmonary vascular endothelium, but suggest that the beta 2 leukocyte integrins CD11/CD18 play only a minor role in this process.


Trauma involving the dentition and supporting tissues.

Successful treatment of traumatic injuries depends on timely action by the patient and a quick and accurate diagnosis by the dentist. Although most injuries are minor and of an urgent nature, displaced or missing teeth are true emergencies. The mismanagement of dental traumatic injuries has provided much information as well as questions for research that have resulted in the increased retention of teeth with as little treatment as possible. Maintaining pulp vitality when possible, utilizing the therapeutic effects of calcium hydroxide, and returning teeth to function as soon as possible are keys to predictable prognosis.


New developments in synthetic bone replacement materials.

Synthetic bone replacement materials continue to be much discussed in the current periodontal literature. Numerous reports have shown their clinical use in the treatment of intraosseous defects. Periodontal treatment aims also include regeneration of a new functional attachment. Although histologic studies have shown that most of the synthetic bone substitutes can enhance bone formation, they are not able to promote new attachment of periodontal tissues to the root surface previously exposed. Future studies are needed to assess whether these materials could be of use together with growth factors in composite grafts or in conjunction with guided tissue regeneration techniques.


Differentiation of mouse embryonic stem cells to lympho-hematopoietic lineages in vitro.

Mouse embryonic stem (ES) cells can differentiate in culture to late stages of many cell lineages. I have found culture conditions that are favorable for development in vitro of ES cells into hematopoietic cells at a stage equivalent to day 11-14 of fetal liver development. I describe here: (1) the growth conditions necessary for maintenance of ES cells in an undifferentiated state, and the conditions that allow differentiation of cystic embryoid bodies that contain precursors of most hematopoietic cell lineages, including lymphoid cells; (2) the development of lymphoid vessels from ES fetuses in vivo; (3) the characterization of lymphoid, erythroid, megakaryoid, and myeloid cells from ES fetuses; and (4) the cloning of cell lines representing lymphoid, myeloid lineage cells from differentiated ES cells.


The amphicrine pancreatic cell line AR42J: a model system for combined studies on exocrine and endocrine secretion.

Secretory vesicles of both the exocrine and the endocrine pancreas have been isolated and characterized in molecular terms from pancreatic tissue and primary cell cultures. Studies on pancreatic secretory processes could be further facilitated by the use of permanent cell lines that respond to secretory stimuli with a regulated secretory response. We now present biochemical, morphological and secretory studies on the rat pancreatic acinar cell line AR42J. This cell line is characterized by the presence of digestive enzyme-containing dense core vesicles, which are released in response to cholecystokinin. In addition, we present evidence that these cells also contain small neuroendocrine-specific vesicles, as evidenced by the expression of the neuroendocrine-specific vesicle proteins synaptophysin and S.V.2. Corresponding to these mixed exocrine-neuroendocrine features, we also found considerable amounts of the neurotransmitters glycine, glutamine and gamma-aminobutyric acid (GABA), as well as the rate-limiting enzyme in GABA synthesis, glutamic acid decarboxylase (GAD) (EC 4.1.1.15) expressed in these cells. We demonstrated a specific uptake mechanism for radioactively-labelled GABA by these cells. In addition, GABA was released from intracellular storage pools by nicotinic receptor stimulation or membrane depolarization. In summary, AR42J cells represent the first amphicrine pancreatic cell line with the combined expression of exocrine and neuroendocrine secretory organelles, both of which follow a regulated secretory pathway in response to various secretory stimuli.


The fetus as an optimal donor and recipient of hemopoietic stem cells.

In the present work we used allogeneic in utero transplantation of fetal stem cells in sheep and monkeys. Thus, both the donor and recipient cells had preimmune status. We showed engraftment of allogeneic stem cells in the tolerant environment of the host. The engrafted cells showed trilineage (lymphoid, erythroid and myeloid) expression of differentiation. Long term maintenance of these engrafted cells was observed. We also demonstrated that ex vivo incubation of donor cells with growth factor can enhance the engraftment. Moreover, we have shown that the engrafted cells respond to phlebotomy in the same manner as endogenous cells. We, therefore, conclude that (a) Preimmune fetuses are highly suitable for stem cell transplantation both as donors and recipients. (b) Engraftment can be modulated by brief maneuvers such as ex vivo manipulation. (c) Functionally, the engrafted cells can respond to hemopoietic stimuli in a similar manner as the endogenous cells. Implications of this transplantation system in clinical medicine is discussed. Everyone who is involved in organ transplantation must, sooner or later, come to grips with immunological barriers which establishes the individuality of each organism by recognizing "self" from "non-self". Transplanters must overcome this barrier, if allogeneic organ transplantation is to be successful. In the case of hemopoietic stem cells (HSC), where immunocompetent cells are transplanted, graft-vs-host disease (GVHD) may also be expected. Tolerance can be expected when the recipient is genetically immunodeficient, thus being unable to mount an immunological barrier.(ABSTRACT TRUNCATED AT 250 WORDS)


Rationale and results of in utero transplants of stem cells in humans.

Following 18 years experience in postnatal fetal liver transplantation (FLT), we have developed a new therapeutical method, namely the in utero transplantation of stem cells from the human fetal liver. This early transplant takes advantage of the immunological tolerance that exists in young fetal recipients. The four fetuses that we treated were 28, 26, 17 and 12 weeks of age (weeks after fecundation). The first two patients had immunodeficiencies, the two other had thalassemia major. Donor cells were obtained from 7- to 12-week-old fetuses, with conditions approved by the National Committee for Bioethics. Donors and recipients were not matched. The fetal cells were infused through the umbilical vein of three patients and injected intraperitoneally into the other one, under ultrasonic visualization. The first patient, bone in 1988, has evidence of engraftment and reconstitution of cell-mediated immunity: initially 10% then 26% of lymphocytes of donor origin (with distinct phenotype), T cell responses to tetanus toxoid and candida antigens. This child, who had bare lymphocyte syndrome, has no clinical manifestation of the disease and lives normally at home. The second child was born in 1989; donor cell engraftment has been proven (Y chromosome in this female patient) and immunological reconstitution is in progress, allowing a normal life at home. The third patient has also evidence of donor cell take (Y chromosome in a female patient) and a partial effect on thalassemia has been documented (donor haemoglobin present in small quantity). In all three cases, no side effect of any kind developed in the mother nor in the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)


Nicotine-induced airway smooth muscle contraction: neural mechanisms involving the airway epithelium. Functional and histologic studies in vitro.

To assess the mechanism of and the role of the epithelium in nicotine-induced bronchoconstriction in vitro, we performed a combined functional and histologic study. Functional study: We suspended tracheal strips or rings from 16 ferrets (1124 +/- 561 g, mean +/- SD) in organ baths. Alternate tracheal strips had their epithelium removed. Dose-response curves to acetylcholine (ACh) and nicotine were established for pairs of tissues with and without epithelium, each pair receiving only one dose of nicotine. Nicotine induced brief muscle contractions not exceeding 25% of the ACh-induced maximum. Contractions were blocked by hexamethonium and 10(-7) M atropine and were abolished or inhibited strongly by tetrodotoxin (TTX), suggesting the involvement of nicotinic neuronal and muscarinic smooth muscle receptors. Removal of the epithelium strongly inhibited contractions at concentrations of nicotine greater than 3 x 10(-5) M which completely removed any dose-response effect. ACh-induced contractions were unchanged, demonstrating smooth muscle integrity. We suggest that the removal of the epithelium attenuates nicotine-induced bronchoconstriction through the removal of nerves running in or close to the epithelium. Histologic study: In tracheae from 15 ferrets (8 male, 7 female), mean weight (+/- SD) 1288 (+/- 470) g, we examined 4 techniques of epithelium removal: (1) gentle scraping with a scalpel blade moved backwards (away from the cutting edge), (2) moving a Q-tip through the unopened tracheal tube without lateral pressure, and (3, 4) stroking the mucosa of opened tracheal segments with a Q-tip, exerting (3) light or (4) moderate pressure. All methods were equally (97%-100%) efficient in removing the epithelium but differed in the amount of damage caused to the basement membrane and/or submucosal tissue. Method (2) caused less damage to the basement membrane than the other methods but still removed almost one-third of it. The study showed that complete removal of the epithelium is at the expense of the submucosa and that a given result of "epithelium removal" is also attributable to removal of the neighboring subepithelial structures.