Role of vitronectin in embryonic rat endocardial cell migration in vitro.

Vitronectin is one of the extracellular matrices that mediate cell spreading and attachment in vitro. In the present paper, we demonstrate the involvement of vitronectin in the migration of cushion mesenchymal cells of the embryonic rat heart. Immunohistochemistry established the localization of vitronectin in the myocardial cells and in some of the cushion mesenchymal cells of the truncus arteriosus and atrioventricular canal. In vitro, vitronectin, fibronectin, and collagen type-I revealed significant stimulating activity for cushion mesenchymal cell migration. The distance migrated by cushion mesenchymal cells cultured on vitronectin, collagen type-I, or both vitronectin and fibronectin was similar, but that on fibronectin was significantly shorter. Following the addition of anti-vitronectin IgG to the medium, the migration distance of cushion mesenchymal cells on fibronectin was remarkably increased. Most explants on vitronectin or on both vitronectin and fibronectin became detached from dishes after the addition of the antivitronectin antibody. Immunostaining revealed that cushion mesenchymal cells cultured on substrata other than vitronectin synthesized vitronectin. From these results, it is suggested that vitronectin is synthesized by myocardial cells and some cushion mesenchymal cells, and that vitronectin inhibits cell movement on fibronectin. This feature of vitronectin may be important in the regulation of the migration of cushion mesenchymal cell in vivo.


[An immunohistochemical and ultrastructural study of 20 chordomas].

Altogether 20 chordomas were reported. The site distribution included 9 cases at the sacrococcygeal region, another 9 cases at the spheno-occipital region, 1 at the cervical vertebra, and another 1 at the lumbar vertebra. Histologic examination revealed that characteristic "physaliphorous cells" were easily identified in all the 19 cases. Tissue for immunohistochemistry study was available in 18 cases. Among them, tumor cells were found strongly positive to EMA, but negative for CEA. 16/18 cases also showed positive for keratin and S-100 protein. Totally, 2 cases were studied ultrastructurally and there were abundant RER and microfilaments seen in the cytoplasm of the tumor cells but only few surface microvilli detected. The epithelial nature of chordoma is strongly supported by the ultrastructural and immunohistochemical findings of these 20 cases.


[Application of microwaves in special and immunohistochemical staining].

Special and immunohistochemical staining are commonly used in clinicopathological diagnosis. However, the processing of most staining methods takes quite a long time even to 1-2 days. Application of microwaves to the staining process such as for nerve myelin, striated muscle fibers, acid mucin, melanin, hemosederin, aspergillus, basement membrane, HHF-35, desmin, cytokeratin, etc. The results were satisfactory similar to those by the ordinary methods and the time required was greatly shortened. Besides, the titres of antibody required were also similar as those used for routine immunohistochemical technique. It is assumed that application of microwaves in staining process might be valuable for pathological diagnosis and particularly the efficiency could be greatly enhanced.


Dissociation between contractile function and oxidative metabolism in postischemic myocardium. Attenuation by ruthenium red administered during reperfusion.

The oxidative metabolic rate may be disproportionately high compared with contractile function in postischemic reperfused myocardium. To study the potential involvement of intracellular calcium transport in high energy expenditure after reperfusion, we determined in isolated rat hearts the effect of ruthenium red, an inhibitor of mitochondrial calcium transport, on recovery of contractile function and oxidative metabolic rate. Hearts subjected to 60 minutes of no-flow ischemia exhibited, at 15 minutes after the onset of reperfusion, poor recovery of left ventricular pressure development to only 7% of the corresponding value measured in control hearts (p less than 0.01). However, myocardial oxygen consumption was recovered to 84% of control (p = NS). The ratio of isovolumic contractile performance (expressed as the product of heart rate and left ventricular pressure development) to myocardial oxygen consumption was severely depressed to 6% of control (p less than 0.01). Supplementation of the perfusate with 6 microM ruthenium red during the initial 40 minutes of reperfusion resulted in a reduction of myocardial oxygen consumption to 65% of the value measured after 15 minutes of reperfusion in hearts reperfused without ruthenium red (p less than 0.01), despite a threefold increase of left ventricular pressure development (p less than 0.05). Oxidation of both palmitate and glucose was reduced to a comparable extent by ruthenium red. The ratio of contractile performance to myocardial oxygen consumption increased progressively during infusion of ruthenium red and did not differ further from control hearts after 30 minutes of reperfusion. Cumulative myocardial release of creatine kinase was reduced by 47% (p less than 0.05) in hearts reperfused with ruthenium red-containing medium. The results provide circumstantial evidence for the hypothesis suggesting that enhanced energy expenditure by intracellular calcium transport may be involved in the mechanisms underlying the dissociation between left ventricular performance and myocardial oxidative metabolic rate early after postischemic reperfusion.


Platelet-derived growth factor isoforms decrease insulin-like growth factor I gene expression in rat vascular smooth muscle cells and selectively stimulate the biosynthesis of insulin-like growth factor binding protein 4.

Platelet-derived growth factor (PDGF) is believed to be a critical mediator of vascular smooth muscle cell (SMC) proliferation. Because insulin-like growth factor (IGF) I (IGF-I) functions as a progression factor for the mitogenic effects of PDGF, we hypothesized that IGF-I gene expression and the production of IGF binding proteins (IGFBPs) by cultured rat aortic SMCs might be regulated by one or more of the three isoforms of PDGF: PDGF-AA, -BB, and -AB. IGF-I gene expression was highly dependent on cell density: IGF-I mRNA transcripts decreased markedly as a function of cell confluence. IGF-I mRNA content was inhibited to a similar degree by PDGF-AA, -BB, and -AB through a mechanism requiring protein synthesis. The inhibition was readily apparent at 4 hours, reaching approximately 25% of control levels after 24 hours. Radioimmunoassayable IGF-I was only barely detectable in SMC-conditioned serum-free medium and not significantly modulated by PDGF. Western ligand blot revealed that vascular SMCs release 30-kd and 24-kd IGFBP into serum-free conditioned medium. PDGF isoforms did not significantly alter release of the 30-kd IGFBP but evoked a fivefold to sixfold increase in the 24-kd IGFBP. The 24-kd IGFBP was found to comigrate with IGFBP-4, a recently identified binding protein that inhibits IGF action. The 30-kd protein was not merely a glycosylated form of IGFBP-4, because it was not sensitive to N-glycanase digestion. PDGF-AA, -BB, and -AB markedly induced expression of IGFBP-4 mRNA in a time- and concentration-dependent fashion. Vascular SMCs also express IGFBP-2 mRNA, but its abundance was not induced by PDGF. In conclusion, PDGF evokes a complex pattern of regulation of genes in the IGF/IGFBP system. By inhibiting IGF-I production and specifically inducing biosynthesis of the inhibitory binding protein IGFBP-4, PDGF may set in motion mechanisms to limit the final magnitude of the mitogenic response.


Inhibition of nitric oxide synthase specifically enhances adrenergic vasoconstriction in rabbits.

1. The effect of inhibition of nitric oxide biosynthesis using N-nitro-L-arginine (NOLA) was examined in conscious rabbits and rabbit isolated aortae. 2. In autonomically blocked conscious rabbits intravenous infusion of NOLA (15 mg/kg) significantly increased arterial pressure and hindlimb vascular resistance but did not affect heart rate. Depressor and hindlimb vasodilator responses to acetylcholine (3-12 micrograms/kg per min) were significantly attenuated in the presence of NOLA. In contrast, NOLA significantly enhanced responses to intravenous infusion of glyceryl trinitrate (10-40 micrograms/kg per min) in vivo. 3. Infusion of noradrenaline (1-4 micrograms/kg per min) or the release of neuronal noradrenaline in response to the infusion of tyramine (80-320 micrograms/kg per min) increased arterial pressure and hindlimb vascular resistance in autonomically blocked conscious rabbits. After the administration of NOLA, the vasoconstrictor responses to both noradrenaline and tyramine were significantly enhanced. 4. In isolated rabbit aortae, NOLA (10 mumol/L) significantly impaired relaxant responses to acetylcholine but did not affect responses to glyceryl trinitrate. NOLA enhanced contractile responses to the adrenoceptor agonists noradrenaline and phenylephrine but did not affect the contractile responses to the thromboxane-mimetic U46619. 5. These data indicate that in autonomically blocked conscious rabbits, NOLA causes systemic vasoconstriction, impairs dilator responses to acetylcholine and enhances dilator responses to glyceryl trinitrate. In addition, NOLA enhances constrictor responses to both exogenous and neuronally-released noradrenaline. These results suggest that nitric oxide is important in the regulation of normal vascular tone and in the modulation of vascular responses to vasodilator and vasoconstrictor agents.


In vitro transcription in isolated nucleoli of Tetrahymena pyriformis.

An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5' region of their 35S pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of transcriptional control during development and differentiation.


Lindane-induced changes in carbohydrate metabolism in Anguilla anguilla.

1. Anguilla anguilla (L.) was exposed to a sublethal concentration of 0.167 ppm (0.25 of the 96-hr LC50) of lindane for 6, 12, 24, 48, 72 and 96 hr. 2. Changes in glycogen, glucose, pyruvate and lactate contents of liver and muscle after lindane exposure, were studied. 3. Muscle and liver glycogen levels decreased significantly during the exposure time. Muscle glucose values increased but on the other hand we found a decrease in those of liver. 4. Muscle and liver pyruvate content increased as did lactate levels in both tissues. 5. The observed effects of lindane on carbohydrate metabolism in fish are discussed in relation to acute stress syndrome.


Substance P in the regulation of mucociliary transport in the frog palate.

1. Mucociliary transport of carbon particles across the palate of anesthetized Rana pipiens was accelerated by electrical stimulation of the palatine nerve. 2. Transport was also accelerated by intracardiac injection of acetylcholine and of substance P in a dose-dependent manner. 3. Acceleration of transport by acetylcholine and by substance P was selectively blocked by atropine and by the substance P antagonist Spantide, respectively. 4. Acceleration of transport by nerve stimulation was blocked partially by atropine and by Spantide, and completely by the two administered concurrently. 5. It is concluded that nerve stimulation releases both acetylcholine and substance P or a similar neuropeptide. 6. Taken in conjunction with the literature, the data suggest that these substances are involved in the normal mechanism for stimulating mucociliary clearance of the palate in response to the presence of particles.


Uridine uptake and RNA synthesis in the brain of torpid and awakened ground squirrels.

1. Uptake of [3H]uridine into the nucleotide precursor pool after intraventricular injection occurs with the same intensity in the brain of torpid and normothermic awakened ground squirrels. This indicates that the membrane uridine transporters and uridine kinases operate in the hibernator's brain in a hypothermia-tolerant way. 2. Utilization of the [3H]uridine pool for synthesis of the rapidly labelled RNA in the brain of torpid ground squirrels falls more than eight times against RNA labelling in the brain of the active animals between bouts of hibernation. 3. Two hours from the beginning of the artificially provoked awakening, RNA uridine incorporation in the brain of ground squirrels has risen 6.5 times. 4. Drastic changes in [3H]uridine RNA labelling under the stable uridine uptake exclude the precursors and energy supply as the main factors determining changes in intensity of the brain RNA synthesis in the different stages of hibernation.


Bactericidal action of a glycoprotein from the body surface mucus of giant African snail.

1. Bactericidal action of a glycoprotein, Achacin, purified from the giant African snail, Achatina fulica Férussac, has been studied. 2. Achacin kills both gram-positive and gram-negative bacteria, but only in their growing states. 3. Achacin does not have any bacteriolytic activity. 4. The strain which has no cell wall is a little more sensitive than the native strain and the cell membrane-damaged strain. 5. Achacin was observed on the cytoplasmic membrane and on the cell wall of treated Escherichia coli by immunoelectron microscopy. 6. Achacin attacks the cytoplasmic membrane of the cell.


Inhibition of RNA- and DNA-synthesis by citrinin and its effects on DNA precursor-metabolism in V79-E cells.

1. The RNA synthesis of V79-E cells was inhibited by the mycotoxin citrinin time- and concentration-dependently. 2. Among the different RNA species mainly the rRNA synthesis was found to be inhibited by 200 microM citrinin. 3. At different precursor concentrations DNA synthesis was inhibited by citrinin after 30 min at least whereas labelling of the acid soluble fractions was found to be 3-fold higher than in untreated cells. 4. Remarkable perturbation of the DNA precursor metabolism, including release of precursor into the medium, was found to occur during citrinin treatment.


Selection and identification of antigenic determinant of Mycobacterium tuberculosis H37Ra strain.

A new procedure for the selection of the antigenic determinant of Mycobacterium tuberculosis H37Ra strain is described. The bacterium components destroyed by ultrasonic were isolated by 15% SDS-PAGE, transferred onto nitrocellulose membrane, immunostained with McAbs against H37Ra. One McAb-positive fragment was obtained using the Western blot analysis. The molecular weight of the antigenic determinant is 35,000 dalton. The antigenic determinant is of immunological reactivity with McAb and TB patients' sera in dot-immunoassay and demonstrated in delayed skin tests in guinea pigs. The amino acid composition of the antigenic determinant was analysed. These suggest that the Western blot analysis may be a useful tool for the analysis of the antigenic determinant.


Identification and structural characterization of an LYT-2/3 homolog in tunicates.

1. A serologic and structural homolog to murine Lyt-2/3 molecular complex was sought in tunicate hemocytes by using a monoclonal antibody specific to Lyt-2 framework determinants (mAb 53-6.7). 2. This antibody labeled a distinct population of tunicate hemocytes, as determined in indirect immunofluorescence and FACS analysis, and immunoprecipitated disulfide-bonded subunits from hemocytes equivalent to the 38 kDa (alpha), 34 kDa (alpha') and 30 kDa (beta) subunits of murine Lyt-2/3 molecules. 3. As in mice, tunicate alpha- and alpha' -subunits each appeared to bear three N-linked oligosaccharides, one high mannose- and two complex-type glycans and focused as a number of heterogeneous spots on IEF gels. 4. In contrast, beta-subunits of both species were associated with a single N-linked glycan of the complex type and focused as basic components of limited charge heterogeneity. 5. Based on tryptic peptide patterns, alpha and alpha' -subunits, are likely to be structurally similar in both tunicate and mouse complexes. 6. However, CNBr cleavage patterns indicated that the alpha-subunit of both species may differ in the size-location of the intrachain disulfide bridge. 7. Collectively our observations suggest the phylogenetic emergence of an Lyt-2/3 homolog at an early level of evolution.


Effect of replacement therapy with cholylsarcosine on fat malabsorption associated with severe bile acid malabsorption. Studies in dogs with ileal resection.

The efficacy of cholylsarcosine, a synthetic deconjugation-resistant and nonsecretory conjugated bile acid analog for the treatment of fat malabsorption caused by severe bile acid malabsorption, was assessed in an animal model. In two dogs, the ileum and ileocecal valve were resected, causing severe diarrhea, steatorrhea, bile acid malabsorption, and progressive weight loss. Cholylsarcosine was administered as the water-soluble sodium salt by mixing with the dog food. Various doses were explored as well as varying intakes of dog food. Fat absorption was assessed by gravimetric measurement of fecal fat; a nonabsorbable recovery marker (polyethylene glycol mol wt 4000) was used to correct for incomplete fecal collections. Cholylsarcosine caused a 5- to 30-fold increase in fat absorption but had no significant effect on weight loss or fecal weight. Duodenal content was collected during digestion of a meal via a surgically placed Thomas cannula; the aspirates were dilute, acidic, and had a low bile acid concentration. The bile acid concentration increased modestly when cholylsarcosine was administered, but remained below the critical micellization concentration. The results indicate that oral administration of cholylsarcosine improved dietary fat absorption in a canine model of severe bile acid malabsorption with associated steatorrhea and bile acid deficiency in the proximal small intestine. Studies with this compound in patients with nutritional problems because of steatorrhea and severe bile acid malabsorption appear warranted.


Clinical evaluation of three anti-HCV ELISAs in patients with various liver diseases.

We measured antibodies to hepatitis C virus (HCV) in 380 patients with various liver diseases by three enzyme-linked immunosorbent assays (ELISAs): HCV antibody ELISA test (C100), KCL-163 (KCL) corresponding to the nonstructural protein of HCV, and JCC based on the translation product of the presumptive HCV core gene. Of 233 cases of non-A, non-B (NANB) liver disease, 63.9% were anti-C100 positive, 69.1% were anti-KCL positive, and 79.8% were anti-JCC positive. Detection of serum HCV-RNA in 213 cases of chronic NANB liver disease revealed that the concordance was 80.3% for C100, 86.4% for KCL, 94.8% for JCC, and 95.3% for all three ELISAs. Overall, 85.4% of chronic NANB cases were considered to have type C disease with HCV infection. The most reliable assays for diagnosing chronic NANB liver disease as type C appeared to be the KCL and JCC ELISAs.


Clarithromycin. A review of its antimicrobial activity, pharmacokinetic properties and therapeutic potential.

Clarithromycin is an acid-stable orally administered macrolide antimicrobial drug, structurally related to erythromycin. It has a broad spectrum of antimicrobial activity, similar to that of erythromycin and inhibits a range of Gram-positive and Gram-negative organisms, atypical pathogens and some anaerobes. Significantly, clarithromycin demonstrates greater in vitro activity than erythromycin against certain pathogens including Bacteroides melaninogenicus, Chlamydia pneumoniae, Chlamydia trachomatis, Mycobacterium chelonae subspecies--chelonae and--abscessus, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium avium complex, Legionella spp. and, when combined with its 14-hydroxy metabolite, against Haemophilus influenzae. However, bacterial strains resistant to erythromycin are also generally resistant to clarithromycin. The antimicrobial activity of clarithromycin appears to be enhanced by the formation in vivo of the microbiologically active 14-hydroxy metabolite. In combination, additive or synergistic activity against a variety of pathogens including Haemophilus influenzae, Moraxella catarrhalis, Legionella species (principally Legionella pneumophila) and various staphylococci and streptococci has been demonstrated. Clarithromycin has a superior pharmacokinetic profile to that of erythromycin, allowing the benefits of twice daily administration with the potential for increased compliance among outpatients where a more frequent regimen for erythromycin might otherwise be indicated. The clinical efficacy of clarithromycin has been confirmed in the treatment of infections of the lower and upper respiratory tracts (including those associated with atypical pathogens), skin/soft tissues, and in paediatrics. Clarithromycin was as effective as erythromycin and other appropriate drugs including beta-lactams (penicillins and cephalosporins) in some of the above infections. A most promising indication for clarithromycin appears to be in the treatment of immunocompromised patients infected with M. avium complex, M. chelonae sp. and Toxoplasma sp. Small initial trials in this setting reveal clarithromycin alone or in combination with other antimicrobials to be effective in the eradication or amelioration of these infections. Noncomparative studies have provided preliminary evidence for the effectiveness of clarithromycin in the treatment of infections of the urogenital tract, oromaxillofacial and ophthalmic areas. However, the promising in vitro and preliminary in vivo activity of clarithromycin against Mycobacterium leprae and Helicobacter pylori warrant further clinical trials to assess its efficacy in patients with these infections. Despite the improved pharmacokinetic profile and in vitro antimicrobial activity of clarithromycin over erythromycin, comparative studies of patients with community-acquired infections reveal the 2 drugs to be of equivalent efficacy. However, clarithromycin demonstrates greater tolerability, principally by inducing fewer gastrointestinal disturbances.(ABSTRACT TRUNCATED AT 400 WORDS)


Pharmacotherapy of childhood asthma. An inflammatory disease.

The recognition that asthma constitutes 2 kinds of physiopathological reactions, namely bronchospasms (immediate reactions) and inflammatory responses (late reactions), suggests that the treatment should be focused against these events. Furthermore, the allergen provocation model, showing the existence of immediate and late asthmatic reactions, can be used to study the effects of different antiasthmatic drugs. Recently, the importance of inflammation in the pathogenesis of asthma in adults has led to the development of therapeutic regimens in which anti-inflammatory treatments are used frequently as a first-line step in the management of asthma. Although at the moment the hard data showing inflammation in childhood asthma are scarce, it is assumed that childhood asthma constitutes the same kind of chronic inflammatory processes as in adult asthma and that its treatment should also include anti-inflammatory drugs.


Drug therapy for nocturnal enuresis. Current treatment recommendations.

It is estimated that enuresis occurs in 5 to 7 million children in the United States. The treatment approach for enuresis is controversial, in large part due to a lack of consensus as to the exact cause of enuresis. Several factors either alone or together may contribute to this syndrome. In addition, there is strong evidence of a genetic component to enuresis. Pharmacotherapy continues to be the preferred treatment for both physicians and families. The most widely used drugs include antidepressants, anticholinergics, and desmopressin. The tricyclic antidepressant imipramine has been used extensively since the 1960s. The exact mechanism of action in enuresis is unknown although it appears to be related to the anticholinergic and antispasmodic effects of the drug. The most common adverse effects reported with imipramine include personality changes, insomnia, anorexia and anxiety. There has been renewed interest in antidiuretic treatment of enuresis. Researchers have found that enuretic children do not have the ability to reduce urine volume at night or concentrate the urine they produce during the night. Clinical trials with desmopressin administered by nasal inhalation report a marked reduction in enuretic episodes. Adverse effects were limited to nasal complaints, rhinitis, or epistaxis. Additional long term studies are needed to delineate desmopressin's role in therapy. Although the number of options for treatment of enuresis is expanding, criteria to predict patient response need to be defined.


Management of gastroenteritis in early childhood.

The most important aspect of modern management of acute diarrhoeal illness in children is that of oral rehydration therapy, and drug therapy is very rarely indicated. Despite the dramatic decline in mortality and morbidity in recent years, there is still the need for continuing education in the appropriate use of oral rehydration solutions. The constitution of oral rehydration solutions and policies of feeding practices during illness continue to be controversial, mainly because of wide variation in the aetiology of the diarrhoea, the nutrition of the child, and the economic and public health factors involved in any particular community. The priority of all healthcare workers is to provide simple guidelines in the use of a safe oral rehydration solution while discouraging unsafe treatments.


Topical corticosteroids. Which drug and when?

A number of factors have to be taken into account when dealing with the treatment of skin diseases with topical corticosteroids, including disease type and phase, potency of the drug, safety, site to be treated, age of the patient, and drug formulation. Concerning the specific indications for topical corticosteroid treatment, we can distinguish skin diseases in which: (a) topical corticosteroids are the treatment of choice; (b) topical corticosteroids are useful as alternative and/or adjuvant treatment; (c) the proposed use of topical corticosteroids has to be confirmed as useful; and (d) topical corticosteroids can be used as a symptomatic treatment. Within these groups, the treatment schedule has to be adjusted according to the above mentioned general factors.


Alglucerase. A review of its therapeutic use in Gaucher's disease.

Alglucerase is a mannose-terminated form of human placental glucocerebrosidase, developed to treat patients with Gaucher's disease. Functional glucocerebrosidase is deficient in Gaucher's disease, an autosomal recessive lipid storage disorder that affects people of all ethnic backgrounds, but has a higher incidence among East European Jews (Ashkenazim). Gaucher's disease manifests with hepatosplenomegaly, bleeding disorders and bone disease, with the more rare subtypes (types 2 and 3) featuring neurological dysfunction. Prior to the development of enzyme replacement therapy, treatment for Gaucher's disease was mainly symptomatic relief. Primary treatment with glucocerebrosidase focuses on removal of the lipid metabolite that causes the pathology. Because of the rarity of Gaucher's disease clinical trials are small, and much of the data investigating alglucerase therapy have been obtained from studies of patients with type 1 disease, the prevalent subtype. Nonetheless, after intravenous administration of alglucerase, improvements are evident within 6 months of therapy. Patients have increased haemoglobin levels and platelet counts, and decreased incidences of epistaxis and bruising. Spleen and liver size are reduced, and skeletal parameters improve. Children gain height and most patients receiving alglucerase therapy are able to resume work and daily activities. Alglucerase is well tolerated, with few mild adverse reactions reported. Although the pharmacokinetic and pharmacodynamic information for alglucerase is limited, its unequivocal efficacy justifies enzyme replacement therapy with this compound as first-line treatment for patients with Gaucher's disease, for whom treatment options are limited.


Didanosine. A review of its antiviral activity, pharmacokinetic properties and therapeutic potential in human immunodeficiency virus infection.

Didanosine is a dideoxynucleoside analogue which undergoes intracellular conversion to the putative active triphosphate metabolite. The active metabolite appears to inhibit viral reverse transcriptase and terminate the proviral DNA, and produces virustatic inhibition of actively replicating human immunodeficiency virus (HIV) at clinically relevant concentrations. In phase I studies didanosine had beneficial effects on various surrogate markers of clinical efficacy and also improved clinical manifestations of HIV infection, with a 21-month survival rate of 80% in patients with acquired immune deficiency syndrome (AIDS) and 93% in patients with AIDS-related complex (ARC) in 1 study. Didanosine also improved CD4+ cell counts in a phase II/III trial in patients previously treated with zidovudine, whereas cell counts declined in patients continuing zidovudine therapy. However, the effects of didanosine on clinical end-points (disease progression, survival, HIV encephalopathy) remain to be established. Peripheral neuropathy and pancreatitis are the predominant dose-limiting adverse events and didanosine therapy should be withdrawn in patients developing signs or symptoms of pancreatitis and during acute treatment of Pneumocystis carinii pneumonia. However, at currently recommended clinical dosages didanosine is generally well tolerated with minimal haematological toxicity. Thus, in a therapeutic area with few treatment options, didanosine offers a welcome alternative for patients intolerant of, or resistant to, zidovudine. There are a number of clinical trials in progress evaluating didanosine alone or in combination with other antiviral agents, and these results are awaited with considerable interest.


The components of merocyanine-540 absorption spectra in aqueous, micellar and bilayer environments.

Spectral-data-processing and curve-fitting techniques have been applied to the decomposition of merocyanine-540 absorption spectra in aqueous, micellar and bilayer environments. The various resolved component bands have been assigned to dye monomers, dimers, or larger aggregates, either in polar or non-polar environments. The analysis of spectral parameters (lambda max and integrated intensity) of the overall spectra and of each component has revealed that merocyanine 540 is a useful probe in studies of membrane structure and dynamics using visible-absorption spectroscopy. In particular, the monomer lambda max and the integrated intensity, i.e. area, of the dimer population are very useful in this respect. The monomer lambda max is especially sensitive to polarity changes and is thus useful, e.g. in the precise determination of critical micellar concentrations. The fractional area of the dimer increases with the packing density of the phospholipid-hydrocarbon region near the interface and is thus very sensitive to changes in vesicle curvature and to the presence of sterols or intrinsic polypeptides in the bilayer.


Intranuclear compartmentalization of transcribed and nontranscribed c-myc sequences in Namalva-S cells.

This investigation is centered on the intranuclear localization of transcribed and nontranscribed c-myc sequences in human Namalva-S cells bearing t(8;14) translocation. Southern hybridization showed that the breakpoint in the truncated allele of c-myc lies outside the characteristic 12.8-kbp EcoRI fragment: as Northern analysis indicated, this reorganization induces a high level of c-myc transcription. Following high-salt treatment, EcoRI digestion and centrifugation, isolated nuclei from the same cells are separated into two residual fractions: a heavier P fraction including nuclear matrix structures and a light S fraction representing dehistonized chromatin fibres. Comparative hybridization experiments revealed that the above procedure separates the c-myc sequences between the two fractions. To locate the site of intranuclear c-myc transcription, we performed run-on experiments with two fractions, topologically analogous to the residual P and S fractions but maintaining the original chromatin organization. These experiments indicated that chromatin P fraction harbours actively transcribed c-myc sequences while chromatin S harbours nontranscribed ones. Further experiments have clarified that the transcribed c-myc sequences are firmly bound to the matrix by multiple attachment sites, arranged throughout the entire gene locus. It was found, moreover, that at the site of attachment the interaction between DNA and the matrix components is realized via proteins. Controls with the beta-globin gene, which is constitutively nonexpressed in Namalva-S cells but upon induction is highly expressed in murine erythroleukemia cells, completely confirmed the conclusion we had made for the intranuclear localization of c-myc. Thus the experiments presented here support the more common idea that the transcribed and nontranscribed sequences are precisely compartmentalized.


Systemically administered antifungal agents. A review of their clinical pharmacology and therapeutic applications.

Systemic antifungal agents express great diversity in their pharmacokinetic profiles, mechanisms of action, and toxicities. Understanding the diverse pharmacokinetic properties of systemic antifungals is critical to their appropriate application. Amphotericin B, drug of choice for most invasive mycoses, has unique pharmacokinetic properties, binding initially to serum lipoproteins and redistributing from blood to tissues. Dosing recommendations are based on the specific infection and the status of the host. Lipid formulations of amphotericin B may be able to attenuate some of its toxicities. Flucytosine is a water-soluble, fluorinated pyrimidine that possesses excellent bioavailability. It is administered only in combination with amphotericin B because of frequent development of secondary drug resistance, and is associated with dose-dependent bone marrow suppression. The antifungal azoles are relatively well tolerated, have broad spectrum antifungal activity, and are fungistatic in vitro. Ketoconazole and itraconazole are highly bound to plasma proteins, are extensively metabolised by the liver, and are relatively insoluble in aqueous solution. By comparison, fluconazole is only weakly bound to serum proteins, is relatively stable to metabolic conversion, and is water soluble. Fluconazole penetrates the cerebrospinal fluid well and is approved for primary and suppressive therapy of cryptococcal meningitis in AIDS patients. The echinocandins have a narrow spectrum of antifungal activity, being effective only against Candida spp.


Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor alpha subunit. Dissecting a potentially myasthenogenic epitope.

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) alpha subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross-reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4-10-residues long were identified. One mAb could bind to either arm on both sides of a beta-turn structure. Five mAb bound to a very-immunogenic cytoplasmic epitope on alpha 373-380 (VICE-alpha). Three of the mAb against VICE-alpha were earlier found to cross-react with non-AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non-myasthenics. Since VICE-alpha has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE-alpha bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE-alpha-like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti-(AChR) mAb in AChR studies.


No evidence for the existence of preformed antibodies against hydroxyethyl starch in man.

Using the highly sensitive ELISA technique for detecting anti-hydroxyethyl starch (HES) antibodies in man sera from 1,056 patients were analyzed. Patients of both sex, who had never had any prior contact with HES, were included in the study. In none of the cases could any titer of HES-reactive antibodies be detected. These data suggest that in man preformed HES-reactive antibodies do not exist or are extremely rare. In any case, unlike dextran-reactive antibodies, they should not have a high clinical importance. The mechanism behind the very rarely observed anaphylactoid reactions after HES application is still unknown.


Efficacy of terazosin in patients with benign prostatic hyperplasia.

Terazosin, a selective, long-acting alpha 1-adrenergic blocker, was evaluated in 44 men with benign prostatic hyperplasia. The dose was titrated from 2 to 20 mg nightly depending on improvement in symptoms and flow rate. All men completed at least 3 months of therapy, 26 had 6 months and 19 received 9-12 months of terazosin. There was an average increase of 2 ml/s in the peak urinary flow rate compared to baseline. This was statistically significant at the 3-month level. Residual urine decreased under treatment at each 3-month time interval. Prior to initiation of terazosin the mean was 165 ml, and it was 62, 100, and 41 ml at 3, 6 and 9 months respectively. There was a statistically significant improvement in both the obstructive and irritative symptom scores. Side effects were minimal; only 1 patient discontinued terazosin due to a hypotensive episode. Terazosin was found to be safe and effective in the dose range of 2-20 mg taken at bedtime in men with symptoms related to benign prostatic hyperplasia. The present study did not identify any baseline parameters such as initial prostate volume, peak flow rates, or obstructive or irritative symptom scores that correlated with clinical outcome.


Nucleolar organizer regions in prostatic adenocarcinomas. Comparison with flow cytometric analysis, tumor grade, stage and serum prostate-specific antigen levels.

Twenty-eight cases of prostatic adenocarcinomas have been selected and the mean argyrophil nucleolar organizer region (AgNOR) count for each case was evaluated. Nine of these tumors were of high, 13 of median and 6 of low differentiation. It was found that as the tumors became less differentiated, the AgNOR count increased (p less than 0.001). Stage of the disease, serum prostate-specific antigen (PSA) value, and DNA ploidy of all these cases have been examined as well but no statistically significant correlation among these three parameters and the AgNOR count was found.


A cytologic, ultrastructural and immunocytochemical comparison of tumor cells and cell cultures originating in invasive bladder carcinoma.

Tumor cells and urine-voided cells from patients with invasive bladder carcinoma as well as from healthy patients were examined cytologically, ultrastructurally and immunocytochemically. The ultrastructure of tumor cells showed an abundant, dilated, rough endoplasmic reticulum in the form of membrane-bound vacuoles full of granular to fibrillar material located perinuclearly and/or paranuclearly. Some cells exhibited enlarged modified lysosomes containing sparce flocculent and particulate precipitate. Papanicolaou staining of these cells showed two basophilic cytoplasmic textures, one green glossy-patchy, perinuclearly and/or paranuclearly, well segregated from the other texture of peripheral hematoxylinophilic foamy cytoplasm, comparable to the cytologic features of cell cultures originating in invasive bladder carcinoma. PAS diastase showed double distribution and texture of the perinuclear glycosaminoglycans, a glossy accumulated mass and large granules. Glycosaminoglycan sacs similar to those of cell cultures were also present in tumor-dispersed cells. There was a nonspecific binding of antisera against lysozyme, human chorionic gonadotropin and alpha 1-trypsin in normal and tumor cells. Tumor cells and tissues were positive for alpha 1-chymotrypsin distributed perinuclearly and in large spheres. Normal cells lacked the above characteristics. The results indicate that it is feasible to use the aforementioned characteristics in conjunction with the existing bladder-cytologic criteria for malignancy as markers in urothelial cancer with regard to prognosis of superficial tumors with high malignant potential.


First Canadian clinical study of transurethral hyperthermia in benign prostatic hyperplasia. Assessment of 220 patients.

Since August 1990, we used the Thermex-II system to treat prostatism in 220 patients for whom an indication of transurethral prostatectomy was proposed, in 85% by some other urologists and the last 15% by us. The treatment was offered on a clinical research basis, and the patient received large information. No complications other than 10 short-term retentions (2-15 days), mild hematuria and 2 marked prostatic edema were noted. Objective amelioration was seen in 78%, and subjective amelioration was noted in 83%. Only 6 patients needed surgery.


Bioavailability of oral isbufylline in rabbits.

The bioavailability of isbufylline was assessed in male rabbits given 12 mg/kg i.v. (intravenous) or per os (oral) according to a randomized design. The concentrations of unbound (fu = 54.0) isbufylline were considered in plasma as a function of time, after i.v. and oral administration. After oral administration in saline solution, a mean absolute oral bioavailability of 59.6% was calculated. The drug is rapidly absorbed and the comparison of the kinetic profiles after i.v. and per os administration, revealed a rapid elimination: t1/2 27.3 and 28.8 min respectively, and a total body clearance of 67.06 ml/min/kg. Urinary recovery 0-48 h accounted for less than 1% of the dose.


Preliminary results of transurethral microwave thermotherapy in the treatment of benign prostatic hyperplasia in Turkey.

This prospective study was conducted in order to evaluate the efficacy of transurethral microwave thermotherapy (TUMT) in the treatment of benign prostatic hyperplasia (BPH). A single TUMT session of 55 min was delivered as an ambulatory procedure without anesthesia to 180 patients. 20 (69%) of the 29 patients who were catheterized due to urinary retention prior to the treatment were able to urinate freely after the treatment. In 91 patients with a follow-up of 3 months, the mean maximum flow rate (MFR) was 7.9 ml/s prior to the treatment and 12.1 ml/s after the treatment (p less than 0.02). The mean residual urine after voiding (PVR) of these patients was 104 and 54 ml before and after TUMT, respectively (p = 0.0001). The mean Madsen symptom score (SS) was 14 before and 7.5 after the treatment (p = 0.0001). The patients with pretreatment MFR higher than 7.0 ml/s, PVR less than 150 ml and SS lower than 15 benefited better from TUMT. As the findings indicated, TUMT is an alternative treatment modality in BPH in selected cases, although the number and the follow-up of our series are insufficient to draw a precise conclusion yet.


Effect of digital rectal examination on serum concentration of prostate-specific antigen.

Prostate-specific antigen (PSA) is used for screening and follow-up of patients with prostate cancer. The effect of certain diagnostic procedures on PSA serum levels is not well defined. Therefore, the effect of digital rectal examination (DRE) on PSA serum levels was investigated. No significant difference was observed in PSA values before and after DRE when blood samples were taken 1-3 min after palpation of the prostate. Kinetic studies demonstrated a significant increase in PSA values up to the factor 3.2 in 14 of 19 patients 2-6 h after DRE. In 9 of 14 cases, PSA was within the initial range 24 h after DRE. That means that a urologist is allowed to take blood samples for the determination of PSA at least within 3 min after palpating a suspicious prostate without getting false-positive results.


Half-life of prostate-specific antigen after radical prostatectomy: the decisive predictor of curative treatment?

Prostate-specific antigen (PSA) half-life calculated in 51 patients subsequent to radical prostatectomy was found to identify patients with residual disease earlier and more reliably than the criterion of not achieving undetectable PSA levels postoperatively. It is proposed that residual tumor affects the half-life by contributing to the serum level of PSA. When PSA half-life was calculated solely in potentially cured patients, we found a half-life of 1.5 days being considerably shorter than in the previous reports based on patient populations regardless of the outcome of disease in the follow-up.


Blockade of thromboxane/endoperoxide receptor-mediated responses in the pulmonary vascular bed of the cat by sulotroban.

The effects of sulotroban (BM13.177; SK & F 95587), a thromboxane (TX) A2/endoperoxide (PGH2) receptor blocking agent on responses to the TXA2/PGH2 mimics, U46619 and U44069, were investigated in the pulmonary vascular bed of the intact-chest cat under constant flow conditions. Injections of U46619 and U44069 directly into the perfused lobar artery caused dose-related increases in lobar arterial pressure without altering left atrial pressure. Following administration of sulotroban in a dose of 5 mg/kg i.v., dose-response curves for U46619 and U44069 were shifted to the right in a parallel manner. The duration of the blocking effect of sulotroban was investigated, and responses to U46619 returned to approximately 50% of control in 120 min and were not significantly different from control 240 min after administration of the receptor antagonist. Sulotroban was without significant effect on responses to prostaglandin (PG) D2 or F2 alpha or serotonin, histamine, norepinephrine, angiotensin II or BAY K8644, an agent which enhances calcium entry. Sulotroban was without effect on responses to endothelin (ET)-1, sarafotoxin (S) 6a or S6c and platelet-activating factor (PAF). Sulotroban did not alter baseline vascular pressures in the cat and responses to the PG and TXA2/PGH2 precursor, arachidonic acid, were reduced. The present data show that sulotroban selectively blocks TXA2/PGH2 receptor-mediated responses in a competitive and reversible manner in the pulmonary vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)


Neurokinin agonists differentially affect A9 and A10 dopamine cells in the rat.

The effect of selective neurokinin (NK) receptor agonists on the activity of A9 and A10 dopamine cells was assessed using extracellular recording. A higher proportion of A10 cells which were administered the NK1 receptor agonist GR73632 or the NK3 receptor agonist senktide showed an effect, whereas the NK2 receptor agonist GR64349 did not discriminate as clearly between the two cell groups. The most frequently encountered response in all cases was an increase in firing rate.


Galanin inhibits sexual behavior in male rats.

Intracerebroventricular injection of galanin potently inhibited (0.5 micrograms/rat) or completely suppressed (5.0 micrograms/rat) copulatory activity in sexually experienced male rats, without producing any other obvious behavioral deficit. It is suggested that galanin, known to potently stimulate feeding behavior, may be involved in the inverse modulation of feeding and sexual behaviors.


Specific binding sites for 125I-endothelin-1 in the porcine and human spinal cord.

Specific binding sites for 125I-endothelin-1 (125I-ET-1) in the spinal cord were investigated using quantitative receptor autoradiographic and chemical cross-linking methods. The binding sites were highly concentrated in porcine and human spinal cord areas corresponding anatomically to the dorsal horn (Rexed's laminae I-III), an area around the central canal (lamina X) and the principal part of the intermediolateral nucleus (IMLp). The localization of the binding sites differed from those of 125I-omega-conotoxin GVIA (125I-CgTx) and 125I-Bolton-Hunter substance P (125I-BH-SP), with the exception that the IMLp shared 125I-ET-1 with 125I-CgTx and 125I-BH-SP binding sites. Specific 125I-ET-1 binding sites in the areas examined were characteristically single and of high affinity. There were no differences between the potencies of unlabeled ET family peptides, ET-1, ET-2, ET-3 and sarafotoxin S6b at inhibiting 125I-ET-1 binding to the areas. Chemical cross-linking studies showed that 125I-ET-1 and 125I-ET-3 mainly bound to a protein with molecular mass of 43 kDa in the porcine and human thoracic spinal cord membranes. The present finding shows the neuronal significance of this newly discovered peptide in the spinal cord.


Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor.

Muscarinic agonists stimulate Cl- secretion across monolayers of the colon tumor epithelial cell line, T84. The muscarinic receptor has been characterized in T84 cell homogenates by radioligand binding using [3H]N-methylscopolamine ([3H]NMS). [3H]NMS bound to a single population of sites at 25 degrees C in 100 mM NaCl, 20 mM HEPES, 10 mM MgCl2, pH 7.4 buffer, with calculated Kd = 278 (+/- 44) pM and Bmax = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide Ki = 0.3 nM) much greater than levetimide (Ki = 8300 nM). Antagonists exhibited the following rank order of potencies and Ki values (nM): atropine (0.54) greater than 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) greater than dicyclomine (14) = hexahydrosiladifenidol (18) greater than pirenzepine (136) greater than AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced 125I efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [14C]arachidonic acid (AA) or [3H]inositol. Carbachol (0.3 mM) did not release [14C]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [14C]AA. Carbachol (0.3 mM) elevated inositol monophosphate 14-fold. The results suggest that muscarinic agonists stimulate Cl- secretion by interacting with an M3 receptor coupled to inositide lipid hydrolysis.


Influence of oligopeptide aldehydes on intracellular Ca2+ concentration in rat pituitary cells.

We investigated the effects of some synthetic tripeptide aldehydes, earlier shown to influence pituitary hormone secretion and 45Ca2+ uptake, on the intracellular free Ca2+ concentration ([Ca2+]i) of rat anterior pituitary cells in suspension. Boc-D-Phe-Leu-Phenylalaninal or Boc-D-Phe-Leu-Prolinal in the tested range of 1-100 or 200 microM, respectively, were ineffective in influencing basal [Ca2+]i but caused a concentration-dependent inhibition in K+ (25 mM)-induced [Ca2+]i elevation. The IC50 of both effects was about 50 microM. In contrast, they did not interfere with the stimulation caused by the calcium channel agonist BAY K 8644 and were also ineffective in influencing the receptor-mediated stimulus of thyrotropin-releasing hormone on [Ca2+]i. On the basis of the present and foregoing results the possible involvement of calcium channels is discussed, but different mechanisms mediating the tripeptide aldehyde inhibition are also considered. A third tripeptide aldehyde, Boc-Gln-Leu-Lysinal (Boc-GLL), showed ionophore-like properties. This nontoxic substance caused a dose-dependent rise up to 400% (at 100 microM) in [Ca2+]i. Its effect is not mediated by voltage-dependent calcium channels, as it cannot be inhibited either by the classical calcium channel antagonists verapamil and nifedipine, or by the above-mentioned inhibitory tripeptide aldehydes. When we decreased the extracellular Ca2+ concentration by the addition of 4 mM EGTA, the effect was inverted and Boc-GLL caused a large fall in [Ca2+]i. We suggest that Boc-GLL may open cell membrane pores through which Ca2+ moves along the concentration gradient.(ABSTRACT TRUNCATED AT 250 WORDS)


Ribosomal RNA gene restriction patterns of Helicobacter pylori: analysis and appraisal of Hae III digests as a molecular typing system.

Ribosomal RNA gene restriction patterns (ribopatterns) of 162 strains of Helicobacter pylori from 93 patients were studied to assess their suitability for use as the basis of a molecular typing system. Computer-assisted numerical analysis of Hae III ribopatterns of 122 strains from 9 countries in 4 continents showed that almost every strain had a distinct and unique ribopattern and only strains from the same individual were genomically matched in all bands or with minor (1-2 band) differences. Hae III ribopatterns offered high typability and reproducibility but were too discriminatory for large-scale epidemiological typing purposes because no rational basis for grouping strains was evident. In contrast, composite band profiles based on 21 band loci within the Hae III ribopatterns, which were used to compare strain sets selected on toxigenicity and geographical origin, were more conserved. Minor differences between some strain sets in several band loci were detected but generally the composite profiles were a reproducible feature of H. pylori. We conclude that Hae III ribopatterns provide an excellent fingerprint for small-scale studies of genomic variation in defined populations, such as sequential patient isolates, but are too specific for general typing of H. pylori.


Characterization of enterococcal isolates by restriction enzyme analysis of genomic DNA.

A restriction enzyme analysis (REA) of chromosomal DNA for the intra-species characterization of enterococci is reported. The DNA was extracted by a rapid method and digested with the restriction enzyme Sal I to provide a characteristic 'fingerprint' consisting of 10-20 bands in the 1.6-5.0 kb range. One hundred and eighty enterococcal isolates were examined; 5 were type strains, 15 from an out-patient clinic and 160 from a geographically isolated British Antarctic Survey Base. The epidemiologically unrelated out-patient clinic isolates gave readily distinguishable patterns, whereas isolates from the geographically isolated community showed evidence of colonization. This technique provided a highly discriminatory method of isolate characterization for Enterococcus faecalis, E. faecium and E. durans suitable for epidemiological studies. A sample of isolates were probed with 16 + 23 S ribosomal RNA from Escherichia coli. Discrimination between isolates was poorer than with REA, although good correlation was observed between the results of the two techniques.


Neuronal loss or replacement in the injured adult cerebral neocortex induces extensive remodeling of intrinsic and afferent neural systems.

The question of how the cerebral cortex responds over time to changes in cortical neuronal number was addressed by inducing excitotoxic cortical neuronal loss, either alone or followed by homotypic fetal cortical cell suspension grafts, in adult rats. Following neuronal cell loss, rapid gliosis and inflammation temporarily maintained tissue volume. As gliosis subsided, tissue shrinkage occurred until the ratio of glia to neurons approached that of normal neocortex. After neuronal loss, total cortical glutamate uptake and glutamic acid decarboxylase activity dropped markedly and remained low, and there was a gradual but considerable reduction in the total size of afferent fiber networks. However, when expressed as concentrations per unit of tissue or protein, histological and neurochemical cortical markers showed increases during the phase of tissue shrinkage and in the long-term equilibrated to near normal levels. Retrograde tracing studies showed that the reduction in total afferent fiber network is accompanied by the atrophy of afferent neuronal cell bodies. Grafts of fetal cortical cells placed after excitotoxic lesions provided long-term reconstitution of cortical tissue mass, maintained afferent fiber systems, and prevented both the atrophy and surrounding cellular gliosis of some afferent neuronal cell bodies for over a year, but were unable to innervate the main cortical target regions in the host. Thus after neuronal loss or replacement, the adult cerebral neocortex and its afferent systems remodel to a density of neurons, glia, and afferent fibers similar to that found in intact tissue, illustrating structural plasticity toward a dynamic equilibrium.


8-Chloro-cyclic adenosine monophosphate, a novel cyclic AMP analog that inhibits human glioma cell growth in concentrations that do not induce differentiation.

Cyclic AMP is supposed to play a role in cell growth and differentiation via activation of protein kinase A. The cAMP signal transduction pathway may therefore be used as a target for the development of anticancer drugs. We compared the effects of 8ClcAMP, a newly developed cAMP analog, to the effects of more commonly used cAMP analogs on the morphology and the proliferation of three human glioma cell lines. 8ClcAMP was the most potent growth inhibitor, exhibiting an IC50 of approximately 10 microM and inducing growth arrest in all three glioma cell lines at a concentration of 100 microM. The cAMP analogs 8CPTcAMP, dibutyryl cAMP, and 8BrcAMP were much less potent. If used in concentrations that induce growth arrest, both 8CPTcAMP and IBMX, but not 8ClcAMP, induced morphological differentiation of the glioma cells. Apparently, the growth-inhibiting effect of 8ClcAMP is not paralleled by its ability to induce morphological differentiation. The explanation for this phenomenon may be that 8ClcAMP does not exert its growth-inhibiting effect via activation of cAMP-dependent protein kinase. Two alternative mechanisms of action are discussed. Since 100 microM 8ClcAMP retarded the growth of normal rat astrocytes only to a marginal extent, without cytotoxic effects, it is concluded that 8ClcAMP may offer interesting perspectives in the treatment of malignant glioma.


Neutrophil kinetics shortly after initial administration of recombinant human granulocyte colony-stimulating factor: neutrophil alkaline phosphatase activity as an endogenous marker.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered (1.5 micrograms/kg body weight) subcutaneously once daily for 5 to 9 days to 5 patients with malignant lymphoma. In all patients, initial administration of rhG-CSF induced a rapid fall in the neutrophil count within 30 minutes, followed by a recovery and an increase in the neutrophil count within 150 min. A rapid fall in the neutrophil count was accompanied by increased expression of neutrophil C3bi-receptors, and neutrophils left in the circulation had lower activity of neutrophil alkaline phosphatase (NAP) and phagocytosis. A decrease in the NAP scores observed at 30 min reflected a preferential decrease of neutrophils with high NAP activity. A recovery and an increase in the neutrophil count were accompanied by a further decrease of NAP scores, which was caused by a preferential increase of neutrophils with lower NAP activity. The NAP scores of mature neutrophils from peripheral blood were not affected by in vitro treatment of cells with rhG-CSF for up to 150 min at 37 degrees C. These findings and the previous observations that neutrophils in the circulating and marginal pools have high NAP activity and neutrophils in the bone marrow pool have low NAP activity taken together suggest that, following initial administration of rhG-CSF, functionally active neutrophils leave the bloodstream preferentially, which is primarily followed by an influx of neutrophils from the bone marrow, but not by demargination of sequestered neutrophils.


Modification of human long-term bone marrow cultures: establishment of a functional stromal microenvironment devoid of myeloid progenitors.

Differences in the plastic adhesive properties of bone marrow (BM) cells were used to initiate modified stromal layers (MSL) from long-term cultures by removing non-adherent cells shortly (4 to 18 hours) after initial seeding. Following this early modification, adherent cells generated a confluent layer after 21 days of culture. Cellular characteristics of volume and spontaneous fluorescence determined by flow cytometry showed that the MSL included 82% fibroblastic stromal cells, 8% macrophages and 10% myelomonocytic cells. Furthermore, clonogenic assays revealed that the MSL were devoid of hematopoietic progenitor cells. MSL were found to sustain long-term myelopoiesis for at least 7 weeks from exogenously added hematopoietic progenitors isolated from bone marrow (CD34+ cells), thereby demonstrating their functionality. The present experimental model appears of interest for the study of interactions between defined populations of hematopoietic cells and cells of the adherent layer. Of importance, our present modifications of human long-term bone marrow culture are technically simple and do not involve manipulation of the stromal cells.


Iron content in human alveolar macrophages.

Intracellular iron can be estimated semi-quantitatively by histochemical determination using the ferrocyanide reagent's score. Particle-induced X-ray emission (PIXE) allows accurate determination of various elements including iron in cells and biological fluids. Both techniques have been used to measure iron in alveolar macrophages gathered by bronchoalveolar lavage. The purpose of this study was to investigate the clinical usefulness of the PIXE technique in occupational respiratory medicine and in various pulmonary diseases. Using the PIXE method, we measured the iron content of alveolar macrophages in healthy subjects, with and without occupational exposure to iron dust, and in patients with pulmonary diseases (chronic obstructive pulmonary disease (COPD), lung cancer, Goodpasture's syndrome). Our results were then compared with those obtained with the ferrocyanide reagent. Intramacrophagic iron was 0.33 +/- 0.21 micrograms.10(-6) (mean +/- SD) cells in healthy non-smoking subjects without occupational exposure. Intramacrophagic iron was increased in smokers, iron-steelworkers, and in patients with COPD or lung cancer even in the absence of pulmonary haemorrhage. The two patients with Goodpasture's syndrome had high intramacrophagic iron content. About 80% of the whole bronchoalveolar lavage fluid iron content was in the cells. Mean iron content of blood monocytes, lymphocytes and neutrophils of eight healthy subjects was significantly lower than that of alveolar macrophages. A significant correlation was found between iron determination by the PIXE method and the ferrocyanide reagent's score (r = 0.89). We conclude that intramacrophagic iron may be increased in steelworkers and subjects with pulmonary haemorrhage, but also in asymptomatic smokers, in COPD and lung cancer patients without occupational exposure to iron dust.


Intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1) expression in the bronchial mucosa of normal and asthmatic subjects.

Bronchial lavage and biopsy studies suggest the involvement of eosinophils and T-lymphocytes in allergic inflammation in asthma. There is evidence suggesting that the expression of adhesion molecules on endothelial cells and of their receptors on leucocytes is involved in this process. To investigate these mechanisms we have obtained bronchial mucosal biopsies from 10 normal subjects and from 10 symptomatic atopic asthmatics. Six of the asthmatics were re-biopsied after 6 weeks of inhaled beclomethasone dipropionate (BDP) during which time their clinical response was monitored. Frozen sections were stained by the immunoperoxidase method using monoclonal antibody (MoAb) 6.5B5 to identify expression of intercellular adhesion molecule (ICAM-1) and MoAb 1.2B6 for endothelial leucocyte adhesion molecule (ELAM-1). Araldite-embedded sections were also stained for eosinophils using MoAb EG2 to identify eosinophilic cationic protein (ECP). A significant mucosal eosinophilia was apparent in the asthmatic but not in the normal biopsies. Immunostaining for ICAM-1 was observed in both the epithelium and endothelium and ELAM-1 in endothelium, with no significant differences being apparent between the asthmatic and normal subjects. Topical BDP markedly reduced the mucosal eosinophilia without affecting the expression of either adhesion molecule. Using this method, we conclude that there is basal expression of ICAM-1 and ELAM-1 in normal human bronchial mucosa, which is not significantly different from that in asthmatics, and that it is insensitive to suppression with corticosteroids at an inhaled dose that causes clinical improvement.


Folylpolyglutamate analogs can inhibit casein kinase II from Xenopus laevis.

Polyglutamate analogs of folate and related compounds were tested as inhibitors of casein kinase II (CK II) obtained from Xenopus laevis. The inhibitory capacity of the pteroyl, 4-amino-10-methyl pteroyl (the methotrexate aromatic moiety), and p-aminobenzoil derivatives increased as the number of gamma-glutamates attached went from 2 to 7. The nature of the aromatic head, group was also important since hexa-gamma-glutamatic acid had no inhibitory activity while the folylhexaglutamate derivatives were strong inhibitors with relative potency of methotrexate greater than pteroyl greater than p-aminobenzoic acid. The inhibition of CK II by methotrexate gamma-pentaglutamate was competitive with casein and showed an apparent K(i) of 90 microM.


The compact state of reduced bovine pancreatic trypsin inhibitor is not the compact molten globule.

Reduced bovine pancreatic trypsin inhibitor (BPTI) has been shown to be in a compact state [(1988) Biochemistry 27, 8889-8893]. This leads to the proposal that this compact state may be a compact molten globule folding intermediate. Optical rotatory dispersion in the visible region failed to show the presence of pronounced secondary structures in the reduced BPTI and no binding of 8-anilino-1-naphthalenesulphonic acid to reduced BPTI could be detected. Yet, no cooperative thermal transition was detected by tyrosine fluorescence. These experiments show that reduced BPTI is not in the compact molten globule state.


Modulation of expression of multidrug resistance gene (mdr-1) by adriamycin.

The acquired resistance to various drugs in cancer is mediated by P-glycoprotein (P-gp) which is encoded by the mdr-1 gene. An increased level of mdr-1/P-gp was demonstrated after chemotherapy administered to treat cancer in humans. To clarify the direct effect of anticancer drugs on mdr-1/P-gp expression, we investigated the change in transport of adriamycin (ADR), and the expression of the mdr-1 gene and P-gp in an ADR-treated, multidrug-resistant leukemic cell line (K562/ADR500). The addition of ADR induced the over-expression of mdr-1/P-gp, which led to a transient decrease in the intracellular accumulation of ADR although the difference was not statistically significant. A maximal effect was observed after 4 h incubation, returning to the baseline level after further incubation for 12-24 h. The phosphorylation of P-gp was inversely correlated with the levels of P-gp. These observations suggest that ADR itself modulates both the expression and function of P-gp. Determination of the optimal schedule for administering adriamycin is essential to achieving the optimal effect in treating cancer.


Influence of chemotherapy on hormone receptor concentration in a xenotransplanted endometrial cancer.

There is growing evidence that chemotherapy may influence the hormone receptor capacity in human carcinomas. The consideration of this assumption may be of importance for the therapeutic management of tumors with metastatic spread which underwent previous adjuvant chemotherapy. Therefore we investigated the influence of six different kinds of chemotherapy on the hormone receptor concentration and the percentage of receptor positive cells in xenotransplanted endometrial cancer. Our results can be summarized as follows: (1) We find neither a significant decrease in hormone receptor capacity after chemotherapeutic treatment (biochemical determination), nor do we see a decrease in the percentage of ER/PR pos. cells (immunohistochemistry). (2) On the other hand, there is no increase in hormone receptor concentration inducible by chemotherapy and no increase in ER/PR pos. cells immunohistochemically.


Hepatitis-C-virus-related chronic liver disease of sporadic type: clinical, serological and histological features.

Hepatitis C virus (HCV) is the most important cause of transfusion-related non-A, non-B hepatitis. It is also thought to be the prime cause of non-transfusion-related or sporadic chronic liver disease. To assess the extent of HCV infection and its significance in this last form, we evaluated the clinical, serological and histological features of 84 consecutive HCV-related patients without a history of blood or blood products transfusion, alcohol or intravenous drug abuse or other known risk factors. Our results indicate that 68 patients (81%) had signs of chronicity, and 33 (39.2%) had superimposed cirrhosis. Serum abnormal alanine aminotransferase and gamma-glutamyltransferase activities represented good predictive markers of liver histological signs of chronicity. The levels of serum gammaglobulins were found to parallel histological severity of liver disease. One or more hepatitis B virus (HBV)-associated markers were present in 52 patients (61.9%). Only 6 (7.1%) were chronic HBV carriers, and 3 of them had signs of active virus replication. These data indicate that HCV plays a major role in the etiology of sporadic chronic liver disease. Its presence is associated with histological forms of chronic liver disease in most patients, who likely represent chronic HCV carriers.


Sensitization against pancreatic antigen in Sjögren's syndrome and chronic pancreatitis. Preliminary communication.

A fractionated pancreatic antigen was prepared using a monoclonal antibody, SP3-1, which reacts with the duct cells of various exocrine organs. The cell-mediated immune response to this antigen was studied by the leukocyte migration inhibition test (LMT) in patients with chronic pancreatitis (CP), Sjögren's syndrome (SjS), and chronic sialoadenitis (CSA). The migration index (MI) for the LMT was 0.97 +/- 0.07 in normal controls (mean +/- SD, n = 11, range: 0.88-1.08). A positive LMT result (MI less than 0.82 = mean -2 SD in controls) was obtained in 7/8 (88%) SjS patients, 4/22 (18%) CP patients, 2/3 CP patients with SjS, and 0/3 CSA patients. These results indicated sensitization against the pancreatic antigen in most SjS patients and some CP patients, and may suggest that an immune response to a common antigenic determinant of the duct cells of exocrine glands plays a role in the pathophysiology of both diseases.


Functional changes of the exocrine perfused rat pancreas in cerulein-induced pancreatitis.

Functional changes of the exocrine pancreas in cerulein-induced pancreatitis were evaluated with the isolated perfused rat pancreas. In control specimens (n = 7), baseline pancreatic juice volume was 0.23 +/- 0.06 microliter/min and after stimulation with CCK-8 (10(-10) M) and secretin (10(-10) M), it was 2.26 +/- 0.45 microliter/min, and in cerulein-induced pancreatitis specimens (n = 8), the corresponding values were 0.11 +/- 0.03 and 0.23 +/- 0.08 microliter/min. The amylase content in the pancreatic juice (IU/min) was 0.73 +/- 0.15 (baseline) and 7.03 +/- 1.66 (stimulated) in the control specimens, and 0.012 +/- 0.002 (baseline) 0.018 +/- 0.004 (stimulated), in the cerulein-induced pancreatitis specimens. Amylase and lipase concentrations in the portal effluents were significantly higher in the cerulein-induced pancreatitis (481.3 +/- 79.4 IU/ml, 283.7 +/- 47.2 BALB U/ml) than in the control specimens (10.7 +/- 1.8 IU/ml, 8.9 +/- 2.9 BALB U/ml). Using the electron microscope fusion of large vacuoles with lateral plasma membrane was observed in cerulein-induced pancreatitis. In cerulein-induced pancreatitis, normal secretion was markedly decreased, and the lateral secretion was suggested to result in the elevation of pancreatic enzyme levels in portal effluents.


Biological characteristics of the Calliphora vomitoria agglutinin.

The galactose specific agglutinin from Calliphora vomitoria was found to be expressed in the haemolymph of all the larval instars, but could not be detected at any other time during the life cycle. The haemagglutinating activity was insensitive to wounding of the tegument or injection of saline; however, a significant increase in haemagglutinating titre could be induced upon inoculation of the haemocoel with biotic or abiotic particulate material. The agglutinin also actively agglutinated several bacterial species and appeared capable of playing a role in particle--haemocyte interaction. The presence of the purified agglutinin significantly increased the attachment of fetuin-derivatized beads to haemocytes in vitro, and this activity could be specifically reduced by the addition of galactose, suggesting that the agglutinin may act as an opsonin.


The effects of retinoic acid on the expression of alpha-fetoprotein and albumin genes in rat hepatoma cell lines.

Many transcriptional regulators can stimulate or repress gene expression depending on the cellular or genetic contexts. Thus dexamethasone increases the amount of alpha-fetoprotein mRNA in Morris rat hepatoma derived cell line McA-RH 8994 cells, but decreases it in McA-RH 7777 cells. In the present study, we demonstrated that retinoic acid, whose receptors belong to the steroid/thyroid hormone receptor gene family, also enhanced the expression of alpha-fetoprotein and albumin gene in McA-RH 8994 cells, but had no effect on alpha-fetoprotein gene expression in McA-RH 7777 cells. In contrast to the effect of dexamethasone on the alpha-fetoprotein gene expression, which requires ongoing protein synthesis, cycloheximide, a protein synthesis inhibitor, enhanced the effect of retinoic acid. Actinomycin D inhibited the retinoic acid mediated increase in alpha-fetoprotein and albumin mRNA expression. Since the McA-RH 8994 cells did not express retinoic acid receptor beta mRNA, the observed regulatory effects of retinoic acid on alpha-fetoprotein and albumin gene expression were not mediated through retinoic acid receptor beta. We also conclude that the regulation was at the level of transcription and that retinoic acid and dexamethasone probably regulate the expression of liver specific genes through different mechanisms.


Sequential induction of syndecan, tenascin and cell proliferation associated with mesenchymal cell condensation during early tooth development.

The cell surface proteoglycan, syndecan, and the extracellular matrix glycoprotein, tenascin, are expressed in the mesenchyme during early development of many organs. We have studied the expression patterns of syndecan and tenascin during initiation of tooth development and in association with mesenchymal cell condensation and compared these with cell proliferation. Syndecan, tenascin and bromodeoxyuridine (BrdU) incorporation were localized by triple-labelling immunohistochemistry in serial sections of molar tooth germs of mouse embryos. Prior to formation of the epithelial tooth bud, syndecan accumulated in the mesenchymal cells which underlie the presumptive dental epithelium, but tenascin was not detected at this stage. Tenascin appeared during initiation of the epithelial down-growth at the lingual aspect of the tooth germ. During subsequent formation of the epithelial bud, at the late bud stage, syndecan and tenascin became exactly colocalized in the condensed mesenchyme which was clearly demarcated from other jaw mesenchyme. The expression of syndecan and tenascin was accompanied by rapid cell proliferation as indicated by marked BrdU incorporation. When development advanced to the cap stage, syndecan staining intensity in the dental papilla mesenchyme increased further whereas tenascin became reduced. In conclusion, the results demonstrate that the expression patterns of syndecan and tenascin overlap transiently during the period of mesenchymal cell condensation and that this is accompanied by cell proliferation. Syndecan and tenascin may play a role in growth control and in compartmentalization of the dental mesenchymal cells in the condensate.


Inhibition of neuronally mediated secretion in rat colonic mucosa by prostaglandin D2.

The effect of prostaglandin D2 (PGD2) on ion transport across the mucosa of the descending colon was studied in rats. PGD2 dose-dependently decreased baseline short-circuit current of mucosa-submucosal preparations mounted either in the Ussing chamber or mounted as an everted sac. However, with the everted sac technique, the tissue was about 1000 times more sensitive to PGD2. Concomitant with the decrease in short-circuit current, PGD2 increased the mucosal-to-serosal fluxes of sodium and chloride and decreased the serosal-to-mucosal flux of chloride. PGD2 inhibited the secretory action of the PGI2 analogue iloprost, PGD2 alpha, and neurotensin. The action of these secretagogues was dependent on the presence of the submucosal plexus. In contrast, PGD2 had no effect on the increase in short-circuit current caused by PGD2, substance P, or serotonin, the actions of which were not dependent on the presence of the submucosal plexus. The results indicate that the action site of the antisecretory mechanism of PGD2 is localized in the secretomotor neurons.


Expression of vascular adhesion molecules in inflammatory bowel disease.

The expression of the vascular adhesion molecules ELAM-1 (endothelial leukocyte adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1) was evaluated in colonic mucosa of patients with inflammatory bowel disease and normal controls by immunocytochemistry. VCAM-1 was found to be constitutively expressed in lymphoid aggregates in normal colonic mucosa and was not significantly enhanced or altered in distribution in mucosa of patients with inflammatory bowel disease regardless of the activity of the inflammatory process. In contrast, ELAM-1 was not detected by these techniques in normal colonic mucosa (n = 11) or in colonic mucosa of patients with inflammatory bowel disease which was either uninvolved or quiescent (n = 30). However, high levels of ELAM-1 were consistently found on endothelial surfaces in association with active inflammation in affected areas of colonic mucosa in patients with either ulcerative colitis (n = 27) or Crohn's colitis (n = 9). In addition, ELAM-1 appeared to be present within neutrophils which had migrated into crypt abscesses in affected mucosa. Similar analysis was carried out in the cotton-top tamarin (CTT), a primate that experiences an idiopathic chronic diffuse colitis resembling human ulcerative colitis. Although anti-human VCAM-1 antibodies did not react with the CTT, anti-human ELAM-1 stained endothelial surfaces in mucosal biopsies from CTT with active colitis. No ELAM-1 was identified in mucosa of CTT in which colitis activity was quiescent. Thus ELAM-1 is expressed on colonic endothelial surfaces in association with inflammation and may play an important role in facilitating leukocyte migration into sites of active IBD involvement.


Identification of motor neurons to the longitudinal muscle of the guinea pig ileum.

Motor neurons that innervate the longitudinal muscle of the guinea pig ileum were identified by retrograde transport from the longitudinal muscle plexus in organotypic culture. Motor neurons had short projections, less than 3.5 mm long, and never had Dogiel type II morphology; most labeled neurons had morphological characteristics of Dogiel type I neurons. Immunoreactivity for choline acetyltransferase was present in 97% of retrogradely labeled nerve cell bodies, reflecting the dominant cholinergic input to the longitudinal muscle layer. Substance P immunoreactivity was present in 48% of motor neurons, indicating that it or a similar tachykinin that mediates noncholinergic excitatory transmission is likely to be released by a subset of cholinergic motor neurons. This strongly suggests that the difference in frequency dependence of substance P and acetylcholine release is attributable to different release mechanisms rather than to activation of separate populations of motor neurons. Immunoreactivity for the calcium-binding protein calretinin was present in 87% of longitudinal muscle motor neurons. The neurochemical coding of longitudinal muscle motor neurons indicated that they constitute about one quarter of all myenteric neurons and are distinct from circular muscle motor neurons.


Somatostatin 28 and coupling of human interdigestive intestinal motility and pancreatic secretion.

To determine the effects of small increases in somatostatin 28 plasma concentrations on human interdigestive gastrointestinal motility and pancreatic secretion, six fasting volunteers were intubated with gastroduodenal multilumen tubes and motility and pancreatic enzyme secretion were measured. Subjects received intravenous NaCl and somatostatin 28 at 11 and 44 pmol.kg-1.h-1 for 120 minutes or at least one interdigestive cycle. The two doses increased plasma somatostatin 28 levels within the physiological or into the supraphysiological range, respectively. Somatostatin 28 at 11 and 44 pmol.kg-1.h-1 decreased the length of the interdigestive motility cycle by 50% and 67% compared with controls, respectively (both P less than 0.002). Propagation velocity of the migrating motor complex (P less than 0.01) and plasma motilin were decreased (P less than 0.01). The smaller and larger dose decreased pancreatic enzyme outputs by 50% and 65%, respectively (P less than 0.005), but with the smaller dose, phase III-associated enzyme outputs were greater than phase I outputs. These findings suggest that small changes in somatostatin 28 plasma concentrations modulate human interdigestive motility and pancreatic enzyme output while coupling of motor and secretory events is preserved.


[Therapy for symptomatic pain in advanced breast cancer].

From 1983-1989, 106 patients with breast cancer were treated in our pain management unit on 6767 treatment days. Pain was caused by bone metastasis in 73% of patients. Neuropathic pain was reported by 32% of the patients. In all but four of these patients, new tumour growth was diagnosed. Patients were treated according to WHO analgesic guidelines with non-opioids on 16% of the days, non-opioids in combination with weak opioids on 36% and with strong opioids on 38% (orally 90%, parenterally 4% of the days). Due to the prevalence of bone pain non-steroidal antiinflammatory drugs were given on 56% of the days. The high incidence of neuropathic pain led to frequent use of co-analgesics (antidepressants 17%, anticonvulsants 12%, steroids 12% of the days). Adjuvant therapy for symptoms other than pain was given on 86% of the days. Whilst 92% of patients reported more than moderate pain on admission, 45% obtained complete pain relief beginning from the first days of treatment. On 92% of the days, patients described their pain as moderate or less. Side effects were treated symptomatically and played a minor role in a reason to change therapy.


Isolation and characterization of a lipopolysaccharide mutant of Legionella pneumophila.

A mutant unable to bind a monoclonal antibody (mAb 1E6) directed against serogroup 1 lipopolysaccharide (LPS) was isolated from L. pneumophila strain Philadelphia-1. SDS-PAGE analysis of isolated LPS from the mutant and wild type revealed that there were no obvious structural differences between the two LPS. The results from Western-blot experiments showed that the mutant LPS was unable to bind mAb 1E6 but retained the ability to bind polyclonal serogroup 1 antibodies. Loss of the LPS epitope recognized by mAb 1E6 did not alter the ability of the mutant to multiply in human monocyte-like U937 cells. Also, the mutant, like wild type, was resistant to killing by normal human serum. These results show that a minor change in the antigenic composition of serogroup 1 LPS has no effect on the virulence properties of strain Philadelphia-1. Additionally, this mutant may be useful for molecular genetic analysis of serogroup 1 LPS biosynthesis and assembly.


Light-activated adenyl cyclase from Trichoderma viride.

The effect of light on adenyl cyclase (E.C. 4.6.1.1) and 3':5'-cyclic-AMP-phosphodiesterase (E.C. 3.1.4.17) activity of Trichoderma viride was investigated. Adenyl cyclase proved to be a membrane-associated enzyme, requiring Mn2+ and was activated by light. In contrast, 3':5'-cyclic-AMP-phosphodiesterase showed no light-stimulated activity. The activity of 3':5'-cyclic-AMP-phosphodiesterase was present mainly in the cytosol and was stimulated by Mg2+.


Bleomycin-ifosfamide-cis-platinum (BIP) in pelvic recurrence of previously irradiated cervical carcinoma: a second look.

The response rate and survival obtained with the combined regimen of bleomycin, ifosfamide, and cis-platinum (BIP) were analyzed in a series of 24 patients with recurrent cervical carcinoma in previously irradiated area. The doses were 30 mg, 5000 mg/m2, and 50 mg/m2, respectively. Mesna was given simultaneously (6000 mg/m2). None of the patients were treated with prior chemotherapy. All the patients were evaluable for toxicity and 20 for response. The median survival in patients evaluable for response was 9 months. No complete and 3 partial responses (15%) were observed, with a median duration of survival of 10+ months (range, 9(+) -16). Stable disease was observed in 8 patients (40%) with a median duration of survival of 9.5 months (range, 6(+) -20). Progressive disease was observed in 9 patients (45%) with a median duration of survival of 6 months (range, 3-25+). Four patients received one course only because of toxicity. One of these patients died at home 6 days after the first course, probably because of dehydration. The main toxicities were myelosuppression, renal impairment, alopecia, and nausea/vomiting. In conclusion, the BIP regimen has considerable toxicity. We were not able to confirm the high response rates earlier reported in pelvic recurrence inside a previously irradiated area. Emphasis in future studies must continue to be placed on the development of more active single agents and combinations.


Paget's disease and melanoma of the vulva. Use of a panel of monoclonal antibodies to identify cell type and to microscopically define adequacy of surgical margins.

The ability of a panel of monoclonal antibodies generated in this laboratory to identify "pagetoid" melanoma cells and distinguish them from true Paget's adenocarcinoma cells in a retrospective analysis of vulvar neoplasms was investigated. Paraffin blocks of formalin and Carnoy's fixed tissue from 15 cases of vulvar Paget's disease and 11 cases of primary vulvar melanoma were retrieved and sections were incubated with the following panel of monoclonal antibodies: HMB45, a melanoma-specific monoclonal antibody; and 35 beta H11 and 34 beta E12, two different anti-cytokeratin monoclonal antibodies, to low molecular and high molecular weight cytokeratins, respectively. The anti-melanoma monoclonal antibody (HMB45) positively identified the melanoma cells, distinguishing them from normal melanocytes, in all 11 cases of melanoma. In contrast, the HMB45 antibody failed to react with the intraepithelial neoplastic cells in all cases of Paget's disease. These latter malignant cells were strongly positive only with the monoclonal anti-low molecular weight cytokeratin antibody 35 beta H11. This latter antibody absolutely distinguished tumor cells from neighboring uninvolved squamous epithelium, which was positive only with the monoclonal antibody 34 beta E12. Using this panel of monoclonal antibodies, the surgical margins could also be better evaluated; in at least one case the surgical margin thought by histological evaluation to be free of tumor was demonstrated by immunocytochemistry to be positive for tumor. In the vulvectomy specimens obtained in both diseases, Paget's or melanoma cells were identified in sections histologically interpreted as free of tumor. Thus, a panel of monoclonal antibodies is able to identify, with high sensitivity and specificity, vulvar melanoma cells and absolutely distinguish them from vulvar Paget's cells and can help in evaluating surgical margins in a more accurate manner.


Orgaran in hip fracture surgery.

Two studies evaluating the effect of Orgaran prophylaxis on the incidence of postoperative thrombosis in hip fracture surgery are reported. In one Scandinavian study, dextran was used in the comparative group, and in the US study, warfarin was used. In both, Orgaran was significantly more effective in reducing the frequency of deep vein thrombosis without producing an increase in bleeding complications or other side effects.


Orgaran (Org 10172): its pharmacological profile in experimental models.

Orgaran is a mixture of glycosaminoglycans extracted from animal mucosa. It consists of heparan, dermatan and chondroitin sulfate; a small proportion of heparan sulfate (4%) has high affinity for antithrombin III (AT III). Orgaran is devoid of heparin or heparin fragments. Orgaran catalyses the inactivation of factor Xa and thrombin. Compared to heparin and most low-molecular-weight heparins, Orgaran has a much higher anti-Xa/anti-IIa ratio. The inactivation of factor Xa is mediated by AT III and that of thrombin by both AT III and heparin cofactor II. Compared to heparin, which is a strong inhibitor of thrombin generation, Orgaran has only moderate inhibitory effects on thrombin generation. Orgaran shows minimal or no effects on platelet function in vitro or in vivo. It inhibits the formation of various types of thrombi (clot-like and mixed thrombi) with approximately the same potency as heparin. Both the high- and low-affinity fraction for AT III contribute to the antithrombotic activity. In contrast to heparin, Orgaran does not inhibit platelet deposition in experimental mixed thrombi unless very high doses of the heparinoid are used. Orgaran is more efficacious than heparin in preventing the extension of established venous thrombosis. Orgaran promotes less bleeding-enhancing activity than heparin in various experimental models. In addition, compared to heparin, it has only minimal effects on platelet degranulation during hemostatic plug formation.(ABSTRACT TRUNCATED AT 250 WORDS)


Anticoagulant mechanisms of Orgaran (Org 10172) and its fraction with high affinity to antithrombin III (Org 10849).

Orgaran (Org 10172), which has antithrombotic activity in man with apparently minimal bleeding side effects, is a mixture of low-molecular-weight heparan, dermatan, and chondroitin sulfates. The degrees to which the minimum concentrations of Orgaran, its fraction with high affinity for antithrombin III (Org 10849; AT III) and unfractionated heparin, which double the activated partial thromboplastin time (APTT) of pooled normal plasma, inhibit intrinsic activation of factor IX, factor X, and prothrombin were compared. Specific ELISAs were used to quantify the activation of each clotting factor. Factor IX activation, which began without a lag phase, preceded factor X and prothrombin activation by approximately 15 and approximately 25 s, respectively. When used at these functionally equivalent concentrations, heparin (2 micrograms/ml plasma), Orgaran (50 micrograms/ml plasma), and Org 10849 (20 micrograms/ml) could delay the onset of factor IX activation. Compared to control plasma, however, only Orgaran reduced the initial rate of factor IX activation. Heparin and Orgaran delayed the onset of factor X activation by 20 and 15 s, respectively, while Org 10849 could not delay the onset of factor X activation. In addition, each anticoagulant delayed the onset of prothrombin activation. Thus, at concentrations which double the APTT of normal plasma, the combined actions of heparan and dermatan sulfate present in Orgaran can apparently suppress factor IX activation more effectively than heparin, and delay the onset of factor X activation nearly as effectively as heparin. The coordinated inhibition of factor IX and factor X activation by Orgaran may contribute to its antithrombotic effectiveness.


Pharmacokinetic considerations on Orgaran (Org 10172) therapy.

Pharmacokinetic investigations on Orgaran (Org 10172) have been conducted by monitoring the following biological effects: plasma anti-Xa, anti-IIa and IIa-generation-inhibiting (IIaGI) activities. In addition, a limited number of studies were conducted on the basis of concentrations of the No-affinity glycosaminoglyc(uron)an (NoA-GAG) fraction as determined by a competitive binding assay. In humans, widely different pharmacokinetic profiles for various biological effects were observed, with relatively short elimination half-lives for the anti-IIa and IIaGI activities of 4.3 +/- 3.5 and 6.7 +/- 3.2 h, respectively, but a relatively long elimination half-life of anti-Xa activity of 24.5 +/- 9.6 h. These differences in half-life mainly reflect differences in the rate of elimination of individual components of Orgaran. Rapid elimination of some of these components may explain why twice daily dosing is required for optimal thrombosis prophylaxis with Orgaran. In a comparative study in healthy male volunteers, the pharmacokinetics of the following low molecular weight heparin(oid)s were determined after intravenous administration: Orgaran (3,750 anti-Xa units), Fragmin (5,000 anti-Xa units), Fraxiparine (7,500 IC units) and Clexane (40 mg). Between these products, wide differences in pharmacokinetics were observed. Particularly, the half-lives of anti-Xa activity and IIaGI activity were much longer for Orgaran than for the other products. At the same time, a relatively low area under the curve of anti-IIa activity was observed. The absolute bioavailability of Orgaran following subcutaneous administration was determined on the basis of plasma anti-Xa and IIaGI activities and the NoA-GAG fraction concentrations. Absorption from subcutaneous tissues was found to be close to 100%, which is significantly higher than of heparin; a finding which indicates that the subcutaneous route is reliable for the administration of Orgaran. The elimination of Orgaran components occurs by renal and possibly non-renal routes. With respect to anti-Xa activity, about 50% of the total clearance can be accounted for by urinary excretion. Therefore, in severe renal failure, a reduction of the maintenance dose of Orgaran would seem to be indicated. Studies on the influence of enzyme induction as a result of treatment with pentobarbital suggest that the pharmacokinetics of Lomoparan are relatively insensitive to changes in hepatic function. In a number of studies, the influence of conditions such as age, body weight and drug interactions were studied. Generally, only minor changes in the pharmacokinetic parameters of Orgaran were observed.(ABSTRACT TRUNCATED AT 400 WORDS)


Orgaran in heparin-induced thrombocytopenia.

Patients who develop heparin-induced thrombocytopenia (HIT) frequently need further anticoagulation to treat an ongoing thromboembolic problem or to prevent one. Orgaran (Org 10172), a low-molecular-weight (LMW) glycosaminoglycuronan, has shown a low frequency (10%) of cross-reactivity in vitro with sera containing the HIT antibody, in contrast to the much higher frequency of cross-reactivity (approximately 80%) shown by the LMW heparins. This paper summarises the results of intravenous or subcutaneous Orgaran treatment in 57 of 67 Australian patients, in whom the diagnosis of HIT was reasonably confirmed by exclusion of other causes of thrombocytopenia and by objective tests. The presenting indications for Orgaran were: continuous venovenous haemofiltration and haemodialysis (n = 21), thrombo-embolism treatment (n = 23), thrombo-embolism prophylaxis (n = 10), and anticoagulation for coronary artery by-pass graft (n = 4), peripheral by-pass graft surgery and plasmapheresis (n = 1 each). The results showed Orgaran to be a safe, well-tolerated, effective (successful treatment in over 90% of patients) anticoagulant in patients with a high thrombotic and/or bleeding risk even if critically ill and requiring haemofiltration. The complete results of the world-wide study in 161 patients confirmed not only these clinical findings in the subgroup of 57 Australian patients, but also the low cross-reactivity (12%) of Orgaran with the HIT serum factor.