Evaluation of an enzyme immunoassay using neoglycolipids constructed from Candida albicans oligomannosides to define the specificity of anti-mannan antibodies. In order to study the respective roles of oligomannoside sequences in the antigenicity of Candida albicans phosphopeptidomannan, a method was developed for constructing neoglycolipids from oligomannosides released by depolymerisation of this molecule. Oligomannosides released by acetolysis were converted to neoglycolipids by coupling them to 4-hexadecylaniline in an equimolar reaction checked by thin layer chromatography. When coated onto microEIA plates, the neoglycolipids exhibited strong reactions which were dose dependent and were saturable with concanavalin A. Reactivity of neoglycolipids with immunoglobulins were then tested with a panel of monoclonal and polyclonal antibodies reacting with epitopes present in the original phosphopeptidomannan. One of two IgM monoclonal antibodies and two of five monospecific rabbit polyclonal IgG reacted strongly with neoglycolipids therefore providing evidence of the presence of structures mimicking epitopes within the pool of neoglycolipids. When 38 sera from 18 hospital inpatients with various levels of antibodies to Candida albicans were tested, a correlation was observed between the EIA to detect neoglycolipids and the EIA to detect phosphopeptidomannan. Successive sera from all patients showing seroconversion in the immunofluorescence assay had increased EIA signals for neoglycolipids.


Treatment of tularemia with ciprofloxacin. A case of tularemia which occurred after close contact with a cat is presented. After unsuccessful amoxicillin treatment, a two-week course of doxycycline was given whereupon the patient responded well. However, the patient relapsed shortly after and was then given ciprofloxacin for two weeks. The patient then recovered completely. Clinical trials are needed in order to establish whether a quinolone could be the drug of choice for treatment of tularemia.


Evaluation of a latex test for rapid detection of pneumococcal antigens in sputum. A latex agglutination test (Directigen) for detection of pneumococcal capsular polysaccharide antigens in sputum was evaluated in comparison to culture and Gram stain using 121 sputum specimens. The sensitivity, specificity and rate of agreement of the latex test compared to culture were 90%, 79% and 81%, respectively. The results suggest that the latex test may be useful for detecting pneumococcal antigen in sputum.


Evaluation of three rapid enzyme immunoassays and cell culture for detection of respiratory syncytial virus. Three rapid enzyme immunoassay techniques for the detection of respiratory syncytial virus antigen (Becton Dickinson Directigen RSV, Abbott RSV Testpack and Abbott RSV EIA) and cell culture were evaluated in a total of 250 nasal washings. The sensitivity and specificity were 62% and 76% respectively for Directigen, 64% and 86% for RSV Testpack, and 76% and 81% for RSV EIA, taking cell culture as the reference method. Agreement between cell culture and EIA techniques was 79% (70 positive and 128 negative results). All three EIA techniques gave positive results in 69 samples (52 positive and 17 negative in the cell culture). In 121 samples all three EIA techniques gave negative results (103 negative and 18 positive in the cell culture). Using the cell culture technique 46 strains other than respiratory syncytial virus were isolated.


Pharmacokinetics of roxithromycin and influence of H2-blockers and antacids on gastrointestinal absorption. The pharmacokinetics of roxithromycin (300 mg orally) and the influence of the antacid aluminum magnesium hydroxide and the H2-blocker ranitidine on bioavailability of roxithromycin in ten healthy volunteers were studied. Pharmacokinetics after a single dose of roxithromycin were characterized by high peak serum levels (9.1 +/- 2.1 mg/l) and a long elimination half-life (7.2 +/- 2.5 h), resulting in a large area under the curve (116.9 +/- 32.7 mg h/l). High inter- and intraindividual variations were found for both the absorption time and the elimination half-life. The bioavailability of roxithromycin was not affected by coadministration with antacids or ranitidine.


Serious complications of vascular catheter-related Staphylococcus aureus bacteremia in cancer patients. Over the period 1986 to 1989, 53 cancer patients were identified with catheter-related Staphylococcus aureus bacteremia at the University of Texas M.D. Anderson Cancer Center. Septic thrombosis was diagnosed in 12 (23%) patients and was suspected in another 3 (6%). Of the 12 patients, five developed deep-seated infections (septic emboli, endocarditis, meningitis, abscess), compared with 2 of the 38 other patients with no septic thrombosis (p < 0.01). Fever persisted for more than three days after antibiotic initiation in 52% of the patients with complications (septic thrombosis and/or deep-seated infections), compared with 19% of those without complications (p < 0.02). Of the three patients with complications who were treated for 14 days with intravenous antistaphylococcal antibiotics, two relapsed; in contrast, all of the nine patients with complications who were treated for more than 14 days (mean 4 weeks) were cured, and none relapsed (p < 0.05). Of the nine patients with complications who were treated with a long course of therapy, only one required surgery. The possibility of septic thrombosis and/or deep-seated infections should be considered in all cancer patients with catheter-related Staphylococcus aureus bacteremia, and if present, the condition should be treated with appropriate intravenous antibiotics for at least four weeks.


Randomized multicenter clinical trial with imipenem/cilastatin versus cefotaxime/gentamicin in the treatment of patients with non-life-threatening infections. German and Austrian Imipenem/Cilastatin Study Group. In an open, controlled, randomized study the safety and efficacy of imipenem/cilastatin was compared with that of the combination cefotaxime/gentamicin (plus metronidazole in patients with suspected anaerobe infection) in the treatment of 337 patients from 12 German and 5 Austrian centers who had non-life-threatening infections. The evaluation was done on an intention-to-treat basis (i.e. all patients including protocol violators) and according to the protocol (144 patients in the imipenem/cilastatin group and 124 in the cefotaxime/gentamicin group). No significant differences were seen between the two treatment groups in terms of the clinical and bacteriological outcome. The frequency of infusion intolerance and thrombophlebitis was low in both groups (< 2%). The overall rate of adverse events was comparable in the two groups, nausea, vomiting and diarrhea being the most frequent events. Nephrotoxicity, indicated by an increase in serum creatinine, was significantly higher in the cefotaxime/gentamicin group. Imipenem/cilastatin was shown to be as effective as cefotaxime/gentamicin (metronidazole) and appears to be well tolerated.


Characteristics of Pseudomonas aeruginosa strains causing septicemia in a Spanish hospital 1981-1990. The epidemiological and biochemical characteristics of Pseudomonas aeruginosa strains causing septicemia in a Spanish hospital over a ten-year period (1981-1990) were analyzed. A total of 207 episodes, corresponding to 0.7 episodes per 1,000 inpatients and 3.2% of the total number of episodes of septicemia, were registered. Males were more often affected than females (rate 3.2:1). The respiratory (24.6%) and urinary (21.2%) tracts were the main portals of entry, while haematologic and solid tumours (15.4%) were the most frequent underlying diseases. More than 86% of the strains were susceptible to ceftazidime, mezlocillin, piperacillin and amikacin. Seventy strains were subjected to typing and analysis of virulence factors. Serotypes O:6, O:11 and O:2 could be considered endemic (each present in more than 11.4% of strains). Pyocin typing, antibiotyping and resistotyping were preferred as secondary typing methods to phage typing and plasmid profile analysis. The combination of methods revealed a large diversity of strains although some clusters predominated. More than 80% of the strains produced several exoenzymes, possessed pyoverdin and showed haemolytic activity, and all except one showed serum resistance. All strains were susceptible to silver and more than 80% to mercury and boron, but all were resistant to iodine.


Evaluation of a chemiluminometric immunoassay for detection of Chlamydia trachomatis in the urine of male and female patients. A chemiluminometric immunoassay (Magic Lite Chlamydia) for detection of Chlamydia trachomatis antigens in first-void urine samples was compared with cell culture using urogenital swabs from 221 men and 242 women. The rate of isolation of Chlamydia trachomatis was 23.5% in men, nearly 80% of whom had symptoms of urethritis, and 8.3% in women, in whom both cervix and urethra samples were tested. In urine sediments from men and women respectively the chemiluminometric assay showed a sensitivity of 80.8% and 70%, a specificity of 97% and 95%, a positive predictive value of 89.4% and 58.3%, and a negative predictive value of 94.3% and 97.2%. Discrepancies between results obtained with the chemiluminometric assay and cell culture were resolved using two polymerase chain reaction techniques to test urogenital samples. The detection of Chlamydia trachomatis in urine samples with the chemiluminometric assay was confirmed to be superior for screening symptomatic men with urogenital infections than women as a lower prevalence population.


Review of nocardial infections in France 1987 to 1990. On the basis of the numbers of Nocardia strains referred to the National Reference Center for Mycoses and Antifungal Agents (NRC), Institut Pasteur, Paris, in the period from 1987 to 1990, it was estimated that between 150 and 250 cases of nocardiosis are diagnosed in France each year. A total of 63 clinical isolates were referred to the NRC and identified as Nocardia asteroides (66.7%), Nocardia farcinica (23.8%), Nocardia brasiliensis (3.2%), Nocardia otitidiscaviarum (4.8%) and Nocardia carnea (1.5%). Nocardia asteroides accounted for 71.4% of pulmonary infections, 80.0% of central nervous system infections and 80.0% of systemic infections. Patients infected with Nocardia farcinica died in 57.1% of cases, compared with 17.6% of patients infected with Nocardia asteroides. Corticosteroid therapy represented a significant factor in mortality. Isolates of Nocardia asteroides revealed variable resistance, whereas isolates of Nocardia farcinica were resistant to most antimicrobial agents. Only amoxicillin/clavulanic acid, imipenem, cefoxitin, kanamycin, amikacin, minocycline and vancomycin showed activity against both species. Nocardiosis caused by Nocardia farcinica may be a growing problem because of the relatively high incidence in AIDS patients and the resistance of this species to most antimicrobial agents.


Quantitative assay for testing susceptibility of HIV isolates to zidovudine and sCD4 (178)-PE40. A quantitative coculture assay is described for determining susceptibility of HIV-1 isolates to zidovudine and sCD4 (178)-PE40 (a 60 kDa hybrid protein consisting of the gp120 binding region of CD4 linked to the translocation and ADP-ribosylation regions of Pseudomonas aeruginosa exotoxin). The assay was relatively simple to perform and gave highly reproducible results often within three to five days. IC50 values of zidovudine for HIV-1 strains isolated from two AIDS patients and an asymptomatic seropositive individual were in the range 0.001-0.002 mumol. Isolates obtained after six months of zidovudine treatment had zidovudine IC50 values of 0.01-0.5 mumol. All isolates were equally sensitive to sCD4 (178)-PE40 (IC50 1-5 micrograms/ml). HIV-1 activation in the chronically infected cell line U-1 was inhibited by sCD4 (178)-PE40 but not by zidovudine.


Clinical evaluation of a new lipid-based delivery system for intravenous administration of amphotericin B. In a retrospective study a new lipid-based delivery system for administration of amphotericin B was evaluated in 26 treatment courses in 22 patients. Amphotericin B was given by infusion diluted in either the lipid solution (1 mg/kg/d; 13 courses) or dextrose (0.92 mg/kg/d; 13 courses). No differences were noted in the serum pharmacokinetics. Fever or rigors were observed in 6 of 13 courses in the conventional amphotericin B group versus none in the lipid amphotericin B group (p = 0.007). Four of 13 courses of treatment were discontinued due to adverse effects in the conventional amphotericin B group compared to none in the other group (p = 0.048). In the lipid amphotericin B group the decrease in creatinine clearance was significantly lower on the fourth day of treatment (p = 0.04) and significantly fewer patients had a decrease of more than 25% in creatinine clearance on the sixth day (4/12 vs 9/11 p = 0.02). These preliminary findings suggest that this lipid amphotericin B formulation is well tolerated with few nephrotoxic effects.


A case of prolonged urinary tract infection caused by Mycobacterium fortuitum. A case of prolonged urinary tract infection caused by Mycobacterium fortuitum in a 56-year old female patient is reported. The infection, which was resistant to therapy with conventional antituberculous agents, responded well to a combination of trimethoprim, sulfamethoxazole and doxycycline.


Effect of subinhibitory concentrations of cefamandole and cefuroxime on adherence of Staphylococcus aureus and Staphylococcus epidermidis to polystyrene culture plates. The ability of cefamandole and cefuroxime to inhibit adherence of staphylococci to polystyrene culture plates was tested in an in vitro assay using eight strains each of Staphylococcus aureus and Staphylococcus epidermidis. The results indicated that subinhibitory concentrations of cefamandole and cefuroxime altered the adherence ability of both staphylococcal species, inhibition of adherence being more marked in the presence of cefamandole. It may be important to consider antiadherence properties in association with bactericidal activity when selecting agents for antibiotic prophylaxis.


Evaluation of a monoclonal antibody for detection of Helicobacter pylori in a direct immunofluorescence test. A monoclonal antibody was developed for detection of Helicobacter pylori in gastric tissue sections in a direct immunofluorescence test. On a comparison of the immunofluorescence test with standard methods for detection of Helicobacter pylori, i.e. culture, the urease activity test and histological examination of tissue sections, using 158 biopsy specimens, 30 specimens were positive in all methods and 64 negative. In the remaining cases comparison was not possible because either immunofluorescence (29 specimens) or the standard methods (16 specimens) gave ambiguous results. The direct immunofluorescence test may have potential as an alternative to standard methods, but further testing in a defined patient population is necessary.


Detection of hepatitis B virus DNA in chronic carriers by the polymerase chain reaction. The polymerase chain reaction (PCR) was used to detect hepatitis B virus DNA (HBV-DNA) in serum samples of 104 chronic HBV carriers. Of 22 patients positive for both HBV surface (HBsAg) and HBVe (HBeAg) antigens, seven were positive for HBV-DNA on dot blot hybridisation, and all 22 positive in the PCR. Of 41 HBsAg positive patients who had antibodies against HBeAg (anti-HBe), only three were positive for DNA-HBV on dot blot hybridisation, however DNA was detected in 30 of them with the PCR. Similarly, of 41 individuals with antibodies against HBsAg (anti-HBs), 23 yielded positive results in the PCR technique, although dot blot hybridisation detected HBV-DNA in only one patient. These results indicate that while serological and conventional DNA hybridisation assays are not sensitive enough to determine the infectivity of HBV chronic carriers, PCR is an accurate method for establishing the status and progression of disease in these patients.


Choroiditis and meningitis in experimental murine infection with Listeria monocytogenes. In a study of central nervous system involvement in experimental listeriosis 27 Swiss CD1 mice were inoculated subcutaneously with Listeria monocytogenes. Systemic infection developed, as shown by the isolation of Listeria monocytogenes and histopathological lesions in the spleen and liver. In the central nervous system a mixed inflammatory infiltration in the ventricular system, especially in the choroid plexus, and leptomeningitis were the most relevant lesions. Inflammatory lesions were associated with the presence of Listeria monocytogenes, as demonstrated by a positive anti-Listeria monocytogenes immunoperoxidase reaction within phagocytic cells. It is suggested that choroiditis and meningitis developed as a consequence of hematogenous dissemination of Listeria monocytogenes within mononuclear phagocytes and penetration of these cells into the ventricular system through the choroid plexus.


In vitro susceptibility of Haemophilus influenzae to cefaclor, cefixime, cefetamet and loracarbef. The susceptibility of 2,212 Haemophilus influenzae isolates cultured in UK clinical laboratories in 1991 was determined for four orally-administered beta-lactam drugs. These isolates included 1,893 ampicillin-susceptible, 191 beta-lactamase-positive and 128 ampicillin-resistant, beta-lactamase-negative Haemophilus influenzae. While 150 (6.8%) isolates were resistant to cefaclor (MIC > or = 16 mg/l) and 85 (3.8%) to loracarbef, all were inhibited by < or = 2 mg/l cefetamet and < or = 1 mg/l cefixime and were therefore susceptible to these agents. Ranges and modes of inhibition zone diameters and MICs indicated that the susceptibility of a variable proportion of the 191 beta-lactamase-positive isolates to cefaclor, loracarbef and cefetamet was reduced compared with the fully susceptible population. In contrast, a major reduction in susceptibility to all four antimicrobial agents was seen among the 128 ampicillin-resistant (MIC 1-64 mg/l) beta-lactamase-negative isolates such that these accounted for 53% and 67% of the total number of organisms resistant to cefaclor and loracarbef respectively. In addition, 23 of 25 isolates inhibited only by > or = 1 mg/l cefetamet and all eight inhibited only by > or = 0.5 mg/l cefixime showed this type of resistance to ampicillin. Results indicate the importance of detecting non-beta-lactamase-mediated resistance to ampicillin and any concomitant diminished susceptibility to other beta-lactam drugs.


Antimicrobial activity of ten macrolide, lincosamine and streptogramin drugs tested against Legionella species. RP59500, a semisynthetic pristinamycin combination, and 14 other antimicrobial agents were tested against 108 Legionella strains. Of the ten macrolide, lincosamine and streptogramin agents tested, the new streptogramin RP59500 ranked seventh in order of activity against Legionella pneumophila on the basis of MIC90 results as follows: clarithromycin = 14-OH clarithromycin (MIC90 0.12 mg/l) > roxithromycin > erythromycin = tylosin = virginiamycin > RP59500 (MIC90 1 mg/l) = azithromycin > dirithromycin > clindamycin (MIC90 8 mg/l). Of all 14 drugs tested in this study, rifampicin was the most potent with an MIC90 of 0.008 mg/l. In this retrospective study of Legionella strains (1981-1990 isolates), we observed no trend toward resistance to the agents investigated.


In vitro antimicrobial activity of the new antibiotic vermisporin. The antimicrobial activity of vermisporin, a new antibiotic produced by fermentation of the fungus Ophiobolus vermisporis, was tested in vitro. Vermisporin inhibited 90% of Bacteroides fragilis and other Bacteroides spp. at 1 microgram/ml (range 0.25-1 micrograms/ml). Clostridium perfringens were inhibited by 1 microgram/ml (range 0.25-2 micrograms/ml). Vermisporin inhibited 90% of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, at 0.5 micrograms/ml (range 0.12-0.5 micrograms/ml). Vermisporin MICs for group A, B, C, F and G streptococci were < 1 microgram/ml when tested in Haemophilus Test Medium but > or = 8 micrograms/ml in the presence of blood. Vermisporin MICs for Enterobacteriaceae, Pseudomonas aeruginosa and Haemophilus influenzae exceeded 64 micrograms/ml. Inhibited organisms had MBCs 16- to 32-fold above the MICs.


In vitro activity of L-627 against anaerobic bacteria. The in vitro activity of L-627 against 370 anaerobic bacterial strains including anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, other Bacteroides spp. and fusobacteria was determined by the agar dilution method. This activity was compared with that of piperacillin, cefoxitin, imipenem, meropenem, clindamycin, metronidazole and chloramphenicol. L-627, imipenem, meropenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested. L-627 had in vitro activity similar to that of the other carbapenems tested.


Ultrasonographic evaluation of Legg-Calve-Perthes disease based on sonoanatomic criteria and the application of new measuring techniques. This study attempts an ultrasonographic classification of Legg-Calve-Perthes disease according to sonoanatomic and sonometric criteria. Four stages, similar to standard radiological staging, could reproducibly be found in 29 patients with unilateral disease. The stages could be significantly differentiated according to descriptive sonoanatomic criteria and partially newly introduced sonographic measurements, including epiphysis-metaphysis-index and acetabulum-epiphysis-distance. Sonographic evaluation of Perthes hips is a simple and standardised procedure, which can serve to stage and monitor the course of the disease and hence reduce the amount of X-ray exposure and treatment costs.


Initial stage of Legg-Calve-Perthes disease: comparison of three-phase bone scintigraphy and SPECT with MR imaging. Twenty-one patients with initial stage Legg-Calve-Perthes (LCP) disease were examined by three-phase bone scintigraphy, single photon emission scintigraphy (SPECT), and magnetic resonance (MR) imaging. On dynamic study, increased activity was present in the epiphysis or the growth plate in 39%; the corresponding figure for blood-pool images was 94%. Pinhole images demonstrated the lateral stripe of revascularization in 57% of patients. Decreased signal intensity in the epiphysis on T1-weighted images proved to be the most sensitive indicator of epiphyseal necrosis on MR imaging and was absent in only 10% of the cases. The subchondral fracture occurred in 62% (including the above 10% of cases), identified by T1-weighted image. Cartilaginous hypertrophy, detected by T1-weighted image, had a high incidence (81%). Joint effusion was identified on T2-weighted images in 90% of patients. The cross-sectional views provided by SPECT and MR imaging permits a better appreciation of the extent of epiphyseal necrosis in LCP disease.


The role of ultrasound and isotope scanning in the management of irritable hips. Patients (n = 181) with the irritable hip syndrome were reviewed. Four of these were found to have Perthes disease and 3 cases had septic arthritis. Ultrasonography provides accurate information as to the presence or absence of an effusion in children with an irritable hip syndrome. The likelihood of a positive result is higher in the early course of the disease process (i.e. within 3 days). Bone scanning, if done routinely will help in the early diagnosis of Perthes disease. Recurrence of the symptoms occurred in 18% of patients and most of them were within 12 months of the first onset of symptoms.


Fracture of the carpal scaphoid: frequency and distribution in a well-defined population. The incidence, location, and type of scaphoid fractures found in a well-defined population is described. Fractures of the carpal scaphoid (n = 442) were identified during an eight-year period, of which 19 (5%) were nonunions. At the initial radiographic examination the fractures were visible on PA views in 70% of the cases (true lateral 10%, scaphoid view neutral 77%, scaphoid view ulnar deviated 73%, and scaphoid view with the X-ray tube tilted 30 degrees distally 71%). Among inhabitants living in the Odense Municipality (population at risk 170648 in 1983 to 174948 in 1989) 222 males and 51 females (age range 9-87 year) sustaining scaphoid fractures during a seven-year were period used for computation of incidences. During the survey, there was an average annual incidence of scaphoid fracture of 8 per 100000 females, and 38 per 100000 males. All patients (except a 9-year-old male) were aged 10 years or over. In the age-group 10-14 years there was an average annual incidence of 3 per 100000 females, and 39 per 100000 males. Average annual incidence per 100000 inhabitants of carpal scaphoid fractures according to the location was proximal 6, middle 15, and distal (fractures of the tuberosity included) 2. Average annual incidence per 100000 inhabitants of carpal scaphoid fractures according to type was transverse 7, horizontal oblique 9, vertical oblique 1, avulsion/fracture of the tuberosity 5, and not stated 1.


High-resolution computed tomography in cystic fibrosis. The sensitivity of high-resolution computed tomography (HRCT) in identifying the pulmonary lesions of cystic fibrosis (CF) was evaluated. Thirty-nine patients (16 males, 23 females; mean age 19.1 years) were examined by chest HRCT. According to Shwachman and Kulczycki criteria, the clinical score of the patients ranged from 40 to 95, thus covering most possible variations of lung disease severity. All the patients presented diffuse thickening of bronchial walls, expression of the characteristic CF bronchial inflammation. Bronchiectases were the second most common lung lesions: discrete dilatation of bronchi was observed in 87% of cases; the localization, pattern and extent of bronchiectasis were accurately detected by HRCT. Pleural thickening and hilar adenopathy were frequently identified (in 64% and 82% of the patients, respectively). Bronchoceles were seen in 64% of the patients; atelectasis (33%) and subpleural bullous dystrophic emphysema (28%) were observed less frequently. On HRCT, the localization of the disease processes within the secondary pulmonary lobule was possible in all patients. In agreement with international literature, the identification of these lesions confirms HRCT as the more sensitive technique for early visualization and location of the manifestations of CF bronchopathy. A larger range of experience coming from a systematic use of HRCT in chronic inflammatory lung diseases would increase our knowledge of pathogenetic processes and allow improvement of therapeutic perspectives.


Mediastinal metastases of infradiaphragmatic malignancies. Mediastinal lymph node metastases from infradiaphragmatic malignancies are reported to be a rare occurrence. The results of previous studies have been based on the evaluation of chest radiographs. In this study we retrospectively reviewed the chest and abdominal computed tomography examinations of 50 such patients. Of these, 25 had renal cell carcinoma and the remainder, other genitourinary or gastrointestinal carcinomas. The major nodal groups involved in the mediastinum were the right paratracheal in 41 cases, subcarinal in 31, paraesophageal in 26, and aortopulmonary in 25. Hilar nodes were involved in 21 cases. In the abdomen, para-aortic and or retrocrural adenopathy was present in 29 cases. Of 21 patients with pulmonary metastases, only 12 cases were associated with ipsilateral hilar lymphadenopathy. Supraclavicular lymphadenopathy occurred in only 8 patients. These results suggest that spread of infradiaphragmatic malignancies to the mediastinum occurs primarily by extension from the retrocrural and para-aortic nodes into the thoracic duct and then by antegrade flow in the thoracic duct and its collaterals, with retrograde flow from these channels to the mediastinal nodes.


Erectile dysfunction: timing of spectral wave-form analysis in the assessment of the function of the cavernosal arteries. Color Doppler ultrasound seems to be an accurate method to evaluate vascular erectile function, in particular the arterial supply of the penis. Because of its relative noninvasiveness and easy performance, it has to precede more invasive evaluation (cavernosography and invasive angiography). Combination of wave form pattern analysis with peak systolic velocity measurements allows differentiation between arterial and venous insufficiency. Fifty-one patients suffering from erectile dysfunction, were examined with use of color Doppler ultrasound. Nine had adequate vascular function with good penile tumescence. However, during these adequate (late) stages of tumescence, peak systolic velocity fluctuated significantly. We conclude that in the late stages of tumescence, velocity measurements are not reliable as a parameter of arterial function.


Sonographic findings in infants with suspected necrotizing enterocolitis. During a three-year period, 27 infants with clinical signs of necrotizing enterocolitis (NEC) underwent ultrasonography; 22 of these infants also had abdominal radiographs within a few hours. Sonographically, portal venous gas (PVG) was seen in 10 and intestinal pneumatosis without PVG in 8 infants. Six children underwent laparotomy within 24 hours after ultrasonography and showed evidence of recent NEC. Three other children, laparotomized weeks later were found to have intestinal strictures as signs of previous NEC. None of the patients died from NEC. Nine infants without abnormal gas distribution did not develop overt NEC. With respect to PVG, sonography is able to support the tentative diagnosis of NEC prior to radiography. However, absence of PVG does not exclude NEC.


Radiographic indicants of adult celiac disease assessed by double-contrast small bowel enteroclysis. The number of folds and lumen diameter of the proximal jejunum and distal ileum were retrospectively measured in 40 double-contrast small bowel enteroclysis studies of patients with a biopsy-proven diagnosis of adult celiac disease (ACD) and in 46 healthy control subjects. For both parameters an inverse radiographic pattern was found in celiacs compared to control subjects, in whom the number of folds and lumen calibers are physiologically greater in proximal jejunum than in the distal ileum. Mean differences in the jejuno-ileal number of folds (-7.1) and lumen calibers (-1.3 cm.) were in fact negative in ACD patients, in whom the values of both the parameters are less in the proximal jejunum than in the distal ileum. Particularly, the sign of "reversal of jejuno-ileal caliber" was both sensitive and specific for diagnosis of ACD in this retrospective series. Double-contrast small bowel enteroclysis can play an important role in excluding or confirming the presence of ACD, provided that an assessment of reproducibility and a prospective re-evaluation of operative characteristics of such radiographic signs are performed.


Computed tomography of retroperitoneal duodenal rupture in blunt abdominal trauma. Two cases of retroperitoneal duodenum rupture following an upper abdominal blunt trauma are reported. Computed tomography (CT) demonstrates at best extraintestinal fluid and air in the retroperitoneum, especially if acquisition in right lateral decubitus is possible. Drawbacks of the method are also reviewed.


Defecography and anorectal manometry.

Defecography and anorectal manometry. In a prospective study anorectal manometry was performed in 160 consecutive patients who were referred for defecography. Leakage of contrast material of standardized viscosity, the anorectal angle (ARA), anal resting pressure (Pr) and squeeze pressure (Ps) were measured and analyzed. This was done to investigate the role of ARA on rectal continence during defecography and to investigate whether relevant findings of manometry can be predicted from findings at defecography as part of the diagnostic work-up in selected patients in order to avoid manometry in these patients. The ARA was of significance in maintaining rectal continence for the contrast medium (P = 0.008). With increasing leakage, Pr and Ps decreased significantly (P less than 0.001). In all fully incontinent patients (n = 36) Ps was distinctly below normal values. Manometry can be avoided in these patients if the level of Pr is not important for therapeutic strategy.


Magnetic resonance imaging of the gastrointestinal tract: investigation of baby milk as a low cost contrast medium. After the incidental observation of the high signal intensity of the upper GI tract in a nourished baby, we tested eight baby milks; five different fresh commercial milks, one sweetened and condensed and two lyophilized milks in order to compare their ability to contrast MR images. The images were obtained with a 1.5 T magnet whereas the "in vitro" water proton relaxation time (T1 and T2) measurements were carried out at 0.5 T. After having selected the most effective lyophilized product, that was prepared according to the manufacturer's instructions, a group of 23 adult patients, 17 males and 6 females, with a mean age of 55.8 years (range 37 to 71 years) were examined. Thirteen patients had gastric cancer and ten patients had rectal or rectosigmoid junction tumors. The most effective imaging sequence was a spin-echo T1.w. After oral intake of milk a good contrast of the stomach, with sufficient distribution in the duodenum and the very proximal bowel, was achieved in all 13 patients with gastric cancer, as was a good depiction of the rectum and the recto-sigmoid junction after enema achieved in the 10 patients with rectal cancers. Disadvantages of lyophilized milk as a contrast agent are due to partial intestinal absorption, inhomogeneous distribution and irregular intestinal passage, whereas a clear advantage of lyophilized milk as a contrast agent is its good acceptance and palatable, inexpensive and non invasive properties. Because of these limitations lyophilized milk cannot be considered a real oral contrast medium but it can enhance MR imaging of the upper abdomen, and mainly of the lower GI tract in infants and adults.


Acute cholecystitis: ultrasonographic staging and percutaneous cholecystostomy. Experience in the treatment of acute cholecystitis with percutaneous cholecystostomy in 29 high-risk and elderly patients is reported. The treatment group included 14 men and 15 women, 21 of whom were over 70 years of age. The suspected clinical diagnosis of acute cholecystitis was confirmed in all cases by ultrasonography (accuracy: 95.6%). The percutaneous cholecystostomy was successful in 27 of 29 cases and these patients had a sudden improvement in their clinical condition; failure of the procedure was due in one patient to dislodgement of the catheter and in another patient to the guide-wire slipping out of the gallbladder. Six patients complained of pain radiating to the right shoulder which disappeared within 30-60 minutes after the procedure. Twenty-three of the 27 patients subsequently underwent elective cholecystectomy. In 22 patients the ultrasonographic findings were compared with the histology; thus enabling us to establish an ultrasonographic staging of acute cholecystitis related to the seriousness of the disease. Percutaneous cholecystostomy is the treatment of choice in high-risk patients, in the elderly, as well as in young patients with impending perforation.


Serious or fatal complications after inadvertent administration of ionic water-soluble contrast media in myelography. The consequences of the inadvertent administration, by the intrathecal route, of ionic contrast media instead of iopamidol in seven subjects are reported. The ionic compounds were diatrizoate, iodamide and ioxitalamate. The outcome was fatal in three out of seven subjects, and it depended on the type and the dose of the administered contrast agent. The serious or fatal reactions observed are a tragic confirmation of the predictive power of neurotoxicity data obtained in animal studies with various iodinated water-soluble compounds. The margin of safety, represented by the ratio of LD50 i.ce. in mice to clinical dose in humans, both normalized to bodyweight, appears to reliably reflect the risk of toxic reactions after intrathecal administration of iodinated contrast agents.


A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment. Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.


Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells. Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.


Presence of Golgi remnant membranes in the cytoplasm of brefeldin A-treated cells. Brefeldin A (BFA) rapidly blocks secretion, induces disassembly of the Golgi complex and causes a redistribution of Golgi components into the endoplasmic reticulum (ER). In addition to these effects on the exocytotic pathway, BFA has been shown to induce fusion of endosomal membranes with the trans-Golgi network in some cell types. To better understand the mechanism through which BFA disrupts the exocytotic traffic, we have examined its effects on the ultrastructural organization of the Golgi complex. Within minutes of exposure to BFA, the Golgi cisternae were fragmented into a number of small tubules and vesicles, many of which had a non-clathrin coat on their cytosolic surface. In addition, a complex structure consisting of anastomosing tubules and associated vesicles appeared in the cytoplasm of cells incubated with BFA for 10 min or longer. These tubular networks were permanent, distinct structures separated from the ER cisternae. They contained cis, middle, and trans Golgi proteins as well as the lipid analogue C5-DMB-ceramide. Furthermore, secretory proteoglycans en route through the Golgi were retained in the lumen of the tubular networks. As judged by the endocytosis of cationized ferritin, endosomes do not contribute to the formation of these tubular networks. Reassembly of the Golgi complex after BFA incubation involved fragmentation and reorganization of the tubular networks as well as fusion with vesicles budded from the ER. We conclude that although in the presence of BFA the bulk of Golgi membranes are induced to fuse with the ER, as indicated by the detection of Golgi markers in this organelle, a fraction of these membranes remain in the cytoplasm organized as Golgi remnants.


Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes. By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.


Cell-adhesive properties of streptavidin are mediated by the exposure of an RGD-like RYD site. The interaction of streptavidin with various cell systems was studied using fluorescent derivatives of the protein. The native unprocessed form of streptavidin bound to cells at low levels and in a nonspecific manner. In contrast, both the truncated "core" streptavidin (the commercially available form) and the biotin-blocked unprocessed protein bound to cells in enhanced levels and in a specific, saturable manner. This suggests that the binding of biotin or cleavage of the terminal portion(s) of the native protein molecule causes conformational changes which lead to the exposure of sites which presumably interact with cell surface receptors. Peptide inhibition studies demonstrated that the majority of binding to cells appears to be dependent on RGD-like specificity, suggesting that the GRYDS sequence of the streptavidin molecule may exhibit such specificity. Indirect immunofluorescence assays revealed that the protein is associated mainly with the cell surface. Moreover, streptavidin was demonstrated to compete with specific monoclonal antibodies to the RGD-binding site on the GpIIbIIIa integrin of activated platelets, thus suggesting that streptavidin may facilitate binding to ubiquitous cell-surface adhesion receptors via RGD mimicry.


Migration of polarized epithelial cells through permeable membrane substrates of defined pore size. We have observed that cells of various epithelial lines exhibit the ability to migrate through permeable membrane substrates containing 3.0 microns pores. Scanning and transmission electron microscopic observations of Vero C1008 and Caco-2 cell lines grown on polycarbonate membranes containing 3.0 microns pores revealed extensive penetration of the filter and the establishment of virtually complete monolayers on the opposing surface. The migration of MDCK cells was also observed to occur under the same conditions; however, the extent of MDCK cell growth on the opposing surface was significantly less than observed for Vero C1008 and Caco-2 cells. Morphological differences were apparent between cells growing on the upper and lower faces of the filter membrane, although cells growing on both surfaces exhibited a polarized phenotype. The cells which invaded the filter were collected and maintained by serial passage. The passaged cells exhibited morphological differences and an altered rate of differentiation in comparison to the parental cell type, suggesting that the invasive cells represent a variant of the parental cell population. Studies using filters of different pore sizes indicated that cellular migration also occurs through pores of 2.0 microns diameter, but not through 1.0 micron (or smaller) pores. These observations have significant implications for studies involving the growth of epithelial cells on permeable membrane substrates containing large pores.


Sea urchin coelomic fluid agglutinin mediates coelomocyte adhesion. The sea urchin Paracentrotus lividus coelomic fluid was found to contain agglutinin which agglutinates animal erythrocytes and promotes adhesion of autologous coelomocytes. Hemagglutinating activity depended upon the presence of calcium ions and was relatively heat-stable. Through a combination of methods including ammonium sulfate precipitation and both size exclusion and ion exchange chromatographies, we purified the anti-rabbit agglutinating factor. The intact agglutinin migrates as a single band with an apparent M(r) of over 200,000. Three distinct protein bands with a calculated M(r) of 174,000, 137,000, and 76,000, respectively were observed under reducing conditions. The purified agglutinin strongly promoted the in vitro adhesion of autologous coelomocytes.


Assembly of a tail-less mutant of the intermediate filament protein, vimentin, in vitro and in vivo. Recent reports on the possible contribution of the non-alpha-helical carboxy-terminal domain ("tail") of type III intermediate filament (IF) proteins to IF assembly have been controversial. To examine the importance and role of this domain, we have therefore engineered a Xenopus laevis vimentin cDNA to code for a tail-less polypeptide and have used it in combination with prokaryotic and eukaryotic expression systems. Here we show that tail-less vimentin, isolated from transfected bacteria (Escherichia coli), when used for assembly in vitro, forms normal-looking, loosely packed IFs. By viscometry we demonstrate that this tail-less vimentin assembles at an even higher rate and into longer IFs than wild-type vimentin. In vivo, i.e., by forced expression in transfected type III IF-free cultured epithelial cells, tail-less vimentin was also recovered in short fibrillar structures, in rodlets and in small as well as large spheroidal aggregates ("granules") that did not reveal any IF substructure. Surprisingly, however, spheroidal aggregate structures formed from the tail-deleted vimentin, were seen not only in the cytoplasm but also in the nucleus, indicating a role of the tail in higher order organization and compartmentalization of the vimentin IF system.


The expression and synthesis of the band 3 protein initiates the formation of a stable membrane skeleton in murine Rauscher-transformed erythroid cells. While the temporal sequences of the synthesis and assembly of membrane skeletal proteins has been studied during erythroid maturation, relatively little is known about the events which initiate the assembly of membrane skeleton at the early stages of mammalian erythroid commitment. To investigate the early events that initiate the assembly of the membrane skeleton in mammalian erythroid cells, we have studied the synthesis and assembly of membrane skeletal proteins in murine Rauscher erythroleukemia virus-transformed cells. These cells are blocked in differentiation at around the early progenitor (burst forming unit-erythroid, BFUe) cell stage but can be induced to differentiate in vitro. Pulse-labeling studies reveal that Rauscher cells actively synthesize alpha spectrin, beta spectrin, ankyrin and band 4.1 proteins. However, the synthesis of the band 3 protein and its mRNA are barely detectable in these cells. The peripheral membrane skeletal components assemble only transiently in the membrane skeleton and turn over rapidly, resulting in about 20-fold lower steady state levels than are found in mature erythrocytes. Upon induction with erythropoietin and dimethyl sulfoxide, the mRNA level and synthesis of band 3 are increased about 50-fold. In contrast, the synthesis of spectrin, ankyrin and band 4.1 is increased only about 1.5 to 2.0-fold. However, after induction, the fraction of these proteins assembled on the membrane is increased, their half-lives on the membrane are nearly doubled with a concomitant 4 to 5-fold increase in their steady-state levels. These results suggest that the synthesis of peripheral membrane proteins is detected at the earliest stages of erythroid commitment and increases only slightly during further differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)


Macromolecular structure of reassembled neurofilaments as revealed by the quick-freeze deep-etch mica method: difference between NF-M and NF-H subunits in their ability to form cross-bridges. Neurofilament (NF) structure and ability to form cross-bridges were examined by quick-freeze deep-etch mica and low-angle rotary-shadow electron microscopy in NFs purified from bovine spinal cord and reassembled in various combinations of NF subunits. When NFs were reassembled from triplet proteins, NF-L, NF-M and NF-H, they were oriented randomly and often fragmented, but their elongated filaments (12-15 nm wide) and the cross-bridges (4-5 nm wide) connecting them were similar in appearance to those of isolated bovine NFs or in vivo rat NFs. Projections extended from the wall of the core filament in almost the same pattern as the cross-bridges and were the same in width and interval (minimum interval, 20-25 nm) as the cross-bridges. Projections were more conspicuous when core filaments were separated by 60 to 80 nm or more, while cross-bridges were more conspicuous when core filaments were close to each other. Projections or cross-bridges extended bilaterally at intervals of 20 to 25 nm where core filaments expanded and formed a network between filaments which were far from one another. When NFs were reconstructed from NF-L alone, only core filaments appeared, the same width as the filaments of triplet NFs. The core filaments were occasionally in almost direct contact with each other, with no projection or cross-bridge. When NFs were reassembled from NF-M alone or NF-L + NF-M, although NF-M core filaments were shorter and slightly thinner than NF-L + NF-M core filaments, both had projections, and both had cross-bridges, but cross-bridges were less evident. Cross-bridges were almost the same in width as those of triplet NFs, but significantly shorter and much less frequent although the minimum interval was the same, and core filaments were not attached to each other. In contrast, when NFs were reconstituted from NF-H alone or NF-L + NF-H, both had conspicuous projections and cross-bridges, similar to those of triplet NFs. Thus, when NFs contained NF-H, they formed frequent cross-bridges and long projections with extensive peripheral branching. When NFs contained NF-M but no NF-H, they tended to form cross-bridges, and to form projections that were shorter and straighter and without peripheral branching. That is, there appears to be a significant difference between NF-M and NF-H in ability to form cross-bridges and thus in interaction with adjacent NFs.


A unique tubulin antiserum attenuates the rate of poleward chromosome movement in anaphase. An antiserum against tubulin, NS20, was previously shown to specifically attenuate both fast axonal transport in vivo (Johnston, K. M. et al., Brain Res. 385, 38-45 (1986)) and in vitro (Johnston, K. M. et al., Cell Motil. Cytoskel. 7, 110-115 (1987)) and flagellar motility (Goldsmith, M. et al., Cell Motil. Cytoskel. 20, 249-262 (1991)). We hypothesized that NS20 blocked motility by binding to a multifunctional motor binding domain on the microtubules (MTs), or axonemes. Here we have examined the effect of microinjecting NS20, at metaphase, into dividing PtK2 cells. Plotting chromosome separation (CS) as a function of time, we report here that CS rates for anaphase A (chromosome-to-pole movement) were reduced by approximately 50% relative to uninjected controls. CS rates for anaphase B (spindle pole elongation) were unaffected by the NS20 antiserum. The inhibition of CS rate during anaphase A by NS20 was significantly greater than the inhibition caused by a control antitubulin serum (PC5). Two possible mechanisms underlying NS20's inhibition of CS during anaphase A were considered. NS20 could block the binding of a kinetochore-associated motor to kinetochore MTs (kMTs) or, alternatively, NS20 could stabilize kMTs against depolymerization. Our results favor the first alternative. In a cold-induced depolymerization assay, NS20 had no selective stabilizing effect on MTs. Moreover, we show that NS20 can selectively block the binding of a well characterized MT-associated motor (kinesin) to MTs, in vitro. These results suggest that NS20 may be defining a unique tubulin binding domain common to the motors underlying vesicle transport, flagellar motility, and chromosome movements during anaphase A.


Evidence for reversible, non-microtubule and non-microfilament-dependent nuclear translocation of hsp90 after heat shock in human fibroblasts. A monoclonal antibody (29A) directed against rat liver heat shock protein M(r) 90,000 (hsp90) was produced. By Western immunoblotting of cytosols prepared from several different tissues and species, 29A was shown to specifically recognize only one band with M(r) approximately 90,000. Localization of hsp90 in human gingival fibroblasts was studied using the 29A antibody by indirect mono- and double-staining immunofluorescence and confocal laser scanning microscopy. The distribution was compared to that of the glucocorticoid receptor (GR) and various cytoskeletal structures. Cells were analyzed in interphase and mitosis under basal culture conditions, after heat shock and after microtubule and microfilament depolymerization, sometimes combined with heat shock. A major part of hsp90 immunoreactivity was diffusely distributed throughout the interphase cytoplasm, but a weak nuclear staining with non-stained nucleoli was also present, however, only detectable after methanol and not after formaldehyde/Triton X-100 fixation. Heat shock induced a time-dependent translocation of hsp90 from the cytoplasm to the cell nucleus reaching a plateau after 15 h. This compartment shift was reversible and also occurred in the absence of intact microtubules or intact microfilaments.


The phosphoinositide pathway of lymphoid cells: labeling after permeabilization by alveolysin, a bacterial sulfhydryl-activated cytolysin. An improved method allowing incorporation of [3H]myo-inositol into the phosphoinositide pool of human lymphoid cells is described. The procedure devised involves cell permeabilization with a thiol-activated membranolytic toxin, alveolysin, and optimization of the phosphoinositide labeling and extraction. In these conditions 4 to 10% of the added [3H]myo-inositol is found intracellularly and half of this amount (2-5%) is incorporated into the phosphoinositide pool in only 1 h as compared to the classical 0.2 to 0.3% incorporation obtained after 10 to 20 h. The integrity of coupling between receptors and phospholipase C was assessed by the inositol phosphate production after cell stimulation by various agonists.


Immunolocalization of secretogranin II, chromogranin A, and chromogranin B in differentiating human neuroblastoma cells. In order to obtain further insights into the expression of the known markers of secretory neuroendocrine dense core organelles, secretogranin II (SgII), chromogranin A (CgA), and chromogranin B (CgB) during neuronal differentiation, the immunolocalization of these proteins was studied by means of double immunofluorescence in both undifferentiated and retinoic acid-differentiated SH-SY5Y human neuroblastoma cells. The majority of undifferentiated cells was not immunolabeled for all three proteins. In the majority of differentiated cells, a clearly punctate SgII immunolabeling indicative of the presence of secretory organelles was present in the Golgi region, at the cell periphery, along the neurites and in growth cones. Only relatively few of the SgII-immunolabeled cells were also immunolabeled for CgA and CgB, and in a single cell the three proteins were not always present in the same organelles. These results, obtained in a cultured cell line, confirm the not necessarily parallel distribution of SgII, CgA, and CgB observed in different neuroendocrine tissues and suggest that SgII may be the best marker of human neuroblastoma cell differentiation.


In vitro differentiation of mouse embryo chondrocytes: requirement for ascorbic acid. Chondrocytes enzymatically dissociated from 13-day-old mouse embryo tibia grow in monolayer culture with a fibroblast-like phenotype and express high levels of type I collagen. Chondrogenesis can be induced by transferring the adherent cells in suspension culture and maintaining them in the constant presence of mouse embryo extract. Round shaping of the cells and formation of multicellular aggregates rapidly follow the passage in anchorage-independent conditions. Cell differentiation is evidenced by a marked decrease in the level of type I collagen and by the induction of type II collagen which accumulates when ascorbic acid is included in the culture medium. The addition of the vitamin also triggers the aggregated chondrocytes to organize their extracellular matrix giving rise to a structure closely resembling the in vivo developing cartilage.


Different proliferative potential of rat and pig hepatocytes in pure primary culture and coculture. In the present study we have compared the growth potential of hepatocytes from rats and pigs and the influence of cocultivation between these hepatocytes and the rat liver epitheloid cell line RL-ET-14. Proliferation, i.e., DNA synthesis, was detected by autoradiography after exposure to [3H]thymidine. Rat hepatocytes cultured at low cell density showed a very low basal growth and responded to epidermal growth factor (EGF) and insulin by a considerable increase in DNA synthesis after 48 h leading to a labeling index (LI) of 33%. Cocultivation with RL-ET-14 cells almost completely blocked the basal as well as the growth factor stimulated proliferation of the rat hepatocytes. In contrast, pig hepatocytes cultured alone showed a much greater growth potential (basal: LI 11%; insulin/EGF:LI 67%) than rat hepatocytes and were further stimulated by cocultivation (basal: LI 39%; insulin/EGF: LI 89%). Density-dependent inhibition of cell growth was less pronounced with pig hepatocytes. Even after reaching confluency, they showed further strong proliferation in pure as well as in cocultures whereas the LI of the rapidly growing clone RL-ET-14 decreased to 40%. Use of conditioned medium from RL-ET-14 cells did not mimic the growth inhibition of rat hepatocytes in coculture indicating that no soluble growth inhibitors produced by the epitheloid cells are responsible for this effect. In particular, the differences between rat and pig hepatocytes in coculture are not simply due to production of TGF-beta by the epitheloid cells since the hepatocytes from both species were inhibited by TGF-beta to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)


The lethal prune/Killer-of-prune interaction of Drosophila causes a syndrome resembling human neurofibromatosis (NF1). The eye color mutant prune (pn) of Drosophila melanogaster shows a lethal interaction with the Killer-of-prune (K-pn) allele of the abnormal wing disc (awd) locus. The awd gene is the Drosophila homologue of the mammalian tumor metastasis gene nm23, and it has been postulated that pn encodes a protein with similarity to a GAP, a GTPase-activating protein. Such GAPs potentially control Ras-like proteins, which are important molecular switches. However, there is only a low sequence homology with the genes for human GAP and neurofibromatosis (NF1), and with yeast IRA1 and IRA2, and there is no evidence for the functional significance of this homologization. I now show that pn mutations lower the concentrations of larval pteridines, and that this phenomenon is enhanced by two orders of magnitude by the lethal interaction between pn and awdK-pn. These gradual effects on the pteridin concentrations indicate a corresponding drop of the pools of free GTP, and favor the involvement of GTP-binding proteins. In addition, cytology reveals a considerable hypertrophy of the neuroglia and the perineurium of the larval brain. Furthermore, the lymph glands of the larvae are highly abnormal and form melanotic (pseudo)tumors upon ageing of the larvae. These pseudotumors consist predominantly of lamellocytes which are part of the cellular defence system of Drosophila. These observations most likely indicate hyperactivity of a Ras-like protein which becomes manifest in cell types equivalent to the cell types affected by human neurofibromatosis (NF1). Thus, it is very suggestive to regard the synthetic lethal system prune/Killer-of-prune as the Drosophila model for human neurofibromatosis.


Improved positron emission tomography quantification by Fourier-based restoration filtering. Positron emission tomography (PET) images are characterized by both poor spatial resolution and high statistical noise. Conventional methods to reduce noise, such as local weighted averaging, produce further deteriorations in spatial resolution, while the use of deconvolution to recover resolution typically amplifies noise to unacceptable levels. We studied the use of two-dimensional Fourier filtering to simultaneously increase quantitative recovery and reduce noise. The filter was based on inversion of the scanner's measured transfer function, coupled with high frequency roll-off. In phantom studies, we found improvements in both "hot" and "cold" sphere quantification. Compared with ramp-only filtering, improvements in hot spot recovery for the highest accuracy filter averaged 13.6% +/- 6.6% for spheres larger than 15 mm; improvements in cold spot recovery averaged 30.7% +/- 4.7%. At the same time, the noise was reduced by a factor of 3 compared with randomly filtering. Fourier-based image restoration filtering is thus capable of improving both accuracy and precision in PET.


Motor stimulation response by technetium-99m hexamethylpropylene amine oxime split-dose method and single photon emission tomography. We applied the technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO) split-dose method in order to evaluate the feasibility of assessing cerebral blood flow (CBF) changes with single photon emission tomography (SPET) during a motor activation task. Eleven normal subjects were studied using the Tomomatic 564 (Medimatic, DK). Five subjects were studied twice at rest and 6 subjects at rest and during a motor task performance (finger opposition movements). A total of 28 mCi of 99mTc-HMPAO was injected in 2 doses with a 1:3 ratio. The first scan was obtained after injection of 7 mCi at rest in all subjects. The second scan was obtained a few minutes later, after injection of the remaining dose (21 mCi), under similar resting conditions or during a motor task performance. The mean brain uptake was proportional to the amount of tracer injected and to the acquisition time for both the first scan (5263 +/- 1266 counts x mCi x min) and the second (5034.4 +/- 966 counts x mCi x min). The grey/white matter ratio was 1.67 +/- 0.019 and 1.67 +/- 0.097 for the two scans, respectively. A three-way analysis of variance (ANOVA) for repeated measure showed no significant effects of side, slice and region of interest (ROI) on the CBF in the 5 subjects studied twice at rest, and the mean regional CBF change was -0.2% +/- 5%.(ABSTRACT TRUNCATED AT 250 WORDS)


Myocardial scintigraphy with iodine-123 phenylpentadecanoic acid and thallium-201 in patients with coronary artery disease: a comparative dual-isotope study. To characterise the clinical usefulness of serial myocardial scintigraphy with iodine-123 phenylpentadecanoic acid (IPPA) in comparison with thallium-201, dual-isotope investigations were performed in 41 patients with angiographically documented coronary artery disease. Both tracers were administered simultaneously during symptom-limited ergometry. Planar scintigrams were acquired immediately after stress, and delayed imaging was performed after 1 h for IPPA and 4 h for 201Tl. Scintigrams were evaluated both qualitatively and quantitatively using a newly developed algorithm for automated image superposition. Initial myocardial uptake of both tracers was closely correlated (r = 0.75, p < 0.001). Both tracers also revealed a similar sensitivity for the identification of individual coronary artery stenoses > or = 75% (IP-PA: 70.0%, 201Tl: 66.3%, P = NS) with identical specificity (69.8%). The number of persistent defects, however, was significantly higher with IPPA (P = 0.021), suggesting that visual analysis of serial IPPA scintigrams may overestimate the presence of myocardial scar tissue. On the other hand, previous Q wave myocardial infarction was associated with a decreased regional IPPA clearance (29% +/- 11% vs 44% +/- 11% in normal myocardium, P < 0.05). The data indicate that serial myocardial scintigraphy with IPPA is essentially as sensitive as scintigraphy with 201Tl for the detection of stress-induced perfusion abnormalities. Quantitative analysis of myocardial IPPA kinetics, however, is required for the evaluation of tissue viability.


Reliability of first-pass radionuclide determination of cardiac output in the upright position at rest and during exercise. The reliability of non-invasive determination of cardiac output using first-pass radionuclide cardiography at rest and during exercise in the upright position was evaluated in 20 patients with coronary artery disease. Cardiac output values ranged from 2.97 to 5.99 l/min at rest and from 5.08 to 10.82 l/min during exercise. Cardiac output results obtained by the radionuclide method were compared with those derived from the thermodilution technique performed simultaneously. The mean difference between the two techniques was 0.02 l/min at rest and -0.34 l/min during exercise; the limits of agreement (mean +/- 1.96 SD) were -1.29 to 1.33 l/min and -1.97 to 1.29 l/min, respectively, indicating an acceptable level of agreement. A high reproducibility of the radionuclide technique was found, with a mean difference between determinations by two observers of 0.03 l/min at rest and 0.21 l/min during exercise, the corresponding limits of agreement being -0.75 to 0.81 l/min and -0.79 to 1.21 l/min, respectively. With the aid of a variance component analysis of two determinations by each of four observers, 95% confidence intervals of +/- 10% at rest and +/- 12% during exercise were computed for the radionuclide cardiac output measurements. The observer variation was most pronounced for the part of the cardiac output determination related to measurement of left ventricular equilibrium activity during exercise. First-pass radionuclide cardiography is a reliable method for determination of cardiac output in cardiac patients at rest and during exercise in the upright position.


The use of technetium-99m hexamethylpropylene amine oxime labelled white blood cells to detect subclinical inflammation of the heart after cardiopulmonary bypass in children with congenital heart disease. Ten children (6 boys and 4 girls, aged 1-9 years old) underwent operations with a cardiopulmonary bypass, and the technetium-99m hexamethylpropylene amine oxine (99mTc-HMPAO) labelled white blood cell (WBC) heart scans were used to detect postoperative leukocyte infiltration in the hearts. The results showed that 80% (8/10) of the cases had subclinical inflammation in the hearts (grading of WBC scans > or = score 2), and a positive correlation (R = 0.77) was noted between the severity of the inflammation (grading of the WBC scans) and the duration of the cardiopulmonary bypass in the operations. Another control group (9 boys and 2 girls, aged 2-13 years old) underwent operations without a cardiopulmonary bypass, and subclinical inflammation of hearts was demonstrated in only 1 case (9%) by the 99mTc-HMPAO labelled WBC scans (grading of WBC scans < score 2) after the operations. There was a significant difference (P < 0.001, by a Wilcoxon rank sum test) based upon the severity of the ischaemic heart damage in the two groups. In our preliminary conclusions, the 99mTc-HMPAO labelled WBC heart scans may provide non-invasive and directly discernible evidence of subclinical inflammation in the heart due to a transient ischaemic state during a cardiopulmonary bypass, even if the clinical symptoms and signs of carditis are not apparent.


Gallbladder response to a second dose of cholecystokinin during the same imaging study. Patients on total parenteral nutrition or after prolonged fasting may require treatment with cholecystokinin (CCK) prior to hepatobiliary imaging. Some may also require evaluation of gallbladder (GB) contractility, and the need for a second dose of CCK may arise. It is not clear whether gallbladder function can be adequately evaluated with CCK when a previous CCK dose had already been administered. We studied ten normal subjects to evaluate GB response to a second CCK injection. The subjects received 20 micrograms/kg sincalide in a 3-min infusion prior to administration of technetium-99m disofenin. They then received an identical sincalide dose at 60 min postinjection, and imaging was continued for another 30 min to quantify GB contraction. Gallbladder ejection fraction (GBEF) values ranged from 42-98% (mean: 71.5 +/- 19%). Pretreatment with CCK does not preclude GB contraction evaluation with a second dose of CCK. Expected GBEF values are similar to those obtained with single CCK injections.


Detection of rhinorrhoea by cisternography in combination with single photon emission tomography, following lumbar injection of iodine-123-labelled albumin. Eight patients were examined. Seven had verified rhinorrhoea and six had had one or more episodes of pneumococcal meningitis. In four of the patients a fistula could not be visualized by computed tomography. All eight patients had injections of iodine-123-labelled albumin by lumbar puncture. Tomoscintigraphy (SPET) was performed 3, 6 and 8-9 h after injection. After 6-8 h we were able to demonstrate a fistula in seven of the patients with verified rhinorrhoea and the findings were confirmed by a subsequent operation. In one of the patients we were not able to visualize any leak of cerebrospinal fluid into the nasal cavity. This patient was not operated upon and we were not able to verify the findings in this patient. The operated patients all did well after the operation with no relapse of meningitis. In conclusion, SPECT-cisternography after intralumbar injection of iodine-123-labelled albumin is a valuable diagnostic tool for the detection of small fistulae in patients with rhinorrhea.


Gamma scintigraphy in the evaluation of pharmaceutical dosage forms. Gamma-scintigraphy is applied extensively in the development and evaluation of pharmaceutical drug delivery systems. It is used particularly for monitoring formulations in the gastrointestinal and respiratory tracts. The radiolabelling is generally achieved by the incorporation of an appropriate technetium-99m or indium-111 labelled radiopharmaceutical into the formulation. In the case of complex dosage forms, such as enteric-coated tablets, labelling is best undertaken by the addition of a non-radioactive tracer such as samarium-152 oxide or erbium-170 oxide followed by neutron activation of the final product. Systems investigated include tablets and multiparticulates for oral administration, enemas and suppositories, metered dose inhalers and nebulisers, and nasal sprays and drops. Gamma-scintigraphy provides information on the deposition, dispersion and movement of the formulation. The combination of such studies with the assay of drug levels in blood or urine specimens, pharmacoscintigraphy, provides information concerning the sites of drug release and absorption. Data acquired from the scintigraphic evaluation of pharmaceutical dosage forms are now being used increasingly at all stages of product development, from the assessment of prototype delivery systems to supporting the product licence application.


Tetralogy of Fallot in three siblings: a familial study and review of the literature. We report a family in which three out of four siblings had tetralogy of Fallot (TOF). The family history showed TOF in the daughter of a maternal cousin, while no other congenital heart diseases were discovered. Although no teratogenic environmental agent was discovered, the absence of parental consanguinity and the presence of another affected relative suggest multifactorial inheritance. Autosomal recessive inheritance cannot be ruled out.


Predictive factors for the effect of gonadotrophin releasing hormone analogue therapy on the height of girls with idiopathic central precocious puberty. The factors influencing the final height of central precocious puberty patients treated with gonadotrophin releasing hormone (GnRH) analogues remain a critical issue. This study compares the predicted final height before and after GnRH analogue therapy to identify predictive factors for final height. Fourteen girls with idiopathic central precocious puberty were treated with a GnRH analogue. All had an active non-regressive form before therapy, full and permanent suppression of oestrogenic activity during therapy (duration greater than 2 years, 3.1 +/- 0.3 years, mean +/- SEM), and the pubertal pituitary-ovarian axis had normalized in all of them 1 year after the cessation of therapy. The mean predicted final height increased from 152 +/- 1.8 cm before therapy to 162.2 +/- 1.2 cm (P less than 0.01) at the last evaluation performed 4.5 +/- 0.3 years after the onset of therapy. The mean gain in predicted final height between the onset of therapy and the last evaluation was 10.2 +/- 1.1 cm. It was correlated with the following data recorded at the onset of therapy: bone age advance over chronological age (r = 0.66, P less than 0.02), predicted final height at the onset of therapy (r = -0.76, P less than 0.001), and the difference between the target height and the predicted height at onset of therapy (r = 0.76, P less than 0.001). We conclude that GnRH analogue therapy is more likely to improve final height prognosis in girls who initially present with a markedly advanced bone age and a great difference between their target and predicted heights. Both these parameters reflect the severity of the disease at diagnosis.


Liver dysfunction and acute cardiocirculatory failure in children. We report 15 children who developed transient liver dysfunction related to hepatic ischaemia. All patients had cardiocirculatory failure 24 h before the onset of liver injury (day 1). Peak serum values of transaminases occurred between day 1 and day 3: SGOT (mean: 759 IU/l, range: 150-4400); SGPT (418 IU/l, 95-2547). Transaminase values decreased rapidly and normalised from day 6 to day 10. Minimum values of prothrombin test (PT) occurred on day 1 (31%, 10-70) and 13/15 patients had a PT less than 50% (27%, 10-44). PT values normalized from day 3 to day 10. Hypoglycaemia was present in 8/15 patients on day 1. Liver dysfunction improved after correction of the circulatory failure. These results confirm that transient hepatic dysfunction, probably as a consequence of hepatic hypoperfusion, may occur frequently in children after acute circulatory failure. We conclude that the diagnosis of ischaemic liver injury or shock liver syndrome in children can be made on clinical and biochemical criteria, and that liver biopsy is unnecessary.


Mediastinal germ cell tumour associated with Klinefelter syndrome. A report of case and review of the literature. A 14-year-old boy with Klinefelter syndrome (KS) and a large mediastinal tumour is presented. Human chorionic gonadotropin and oestradiol were markedly increased. An attempt at radical resection was performed. Histological examination revealed a malignant germ cell tumour of mixed histologic pattern composed of choriocarcinoma and components of mature teratoma. Four courses of cisplatin, bleomycin, and etoposide were given. The patient is without any evidence of tumour recurrence 20 months after diagnosis. A review of the literature revealed another 40 cases of primary mediastinal germ cell tumour (PMGCT) associated with KS. Compiled data from larger series demonstrate that at least 8% of male patients with PMGCT have KS, 50 times the expected frequency. In contrast to PMGCT in patients without KS, all tumours were of nonseminomatous histology, and the average age was considerably lower, Tumours in prepubertal boys were associated with precocious puberty.


Polymerase chain reaction-amplification of urease genes: rapid screening for ureaplasma urealyticum infection in endotracheal aspirates of ventilated newborns. Ureaplasma urealyticum infection has been considered to play an important role in the development of bronchopulmonary dysplasia (BPD) in premature infants. Since standard culture methods of U. urealyticum are difficult to perform, new rapid and sensitive methods are needed to detect lung infection of ventilated newborns. Here we describe the polymerase chain reaction as a rapid method to screen endotracheal aspirates for ureaplasma infection. Urease-specific sequences could only be detected in 1 out of 36 ventilated newborns. The procedure described in this paper may facilitate further studies to determine the role of U. urealyticum in development of BPD.


Tick-borne encephalitis in a 3-month-old child. Tick-borne encephalitis has not been reported in infants younger than 12 months of age. We report a 3.5-month-old child with a serologically proven tick-borne encephalitis. The infant had a history of a tick bite 3.5 weeks before the first symptoms of encephalitis appeared. The family lives in an endemic area of the disease. There were no prodromal signs and the course of the disease was monophasic. In an endemic area, prophylactic treatment with hyperimmunoglobulin after a tick bite should be considered even in very young infants, but in most children active immunization is probably not necessary because of infrequent exposure. Active immunization is still recommended after the 1st year of life.


Unsuspected rheumatic heart underlying group B streptococcal endocarditis at the age of 20 months. The case is reported of a 20-month-old girl admitted to our centre for group B streptococcal endocarditis who died of complete atrioventricular block after a week of treatment. There was no history pointing to the presence of a heart disorder. Necropsy disclosed the surprising existence of a vast rheumatic carditis involving essentially the aortic and mitral valves, with bacterial superinfection of the former. The double rheumatic and infectious lesion in such a young patient with infective pseudo-aneurysms of the sinuses of Valsalva makes this an exceptional case.


Pulmonary hypertension complicating a ventriculo-atrial shunt. The use of ventriculo-atrial shunts for CSF diversion in developing hydrocephalus has become rare because of the risk of chronic thrombo-embolism and subsequent pulmonary hypertension associated with an unfavourable prognosis. However, there remains a group of patients in whom ventriculo-atrial shunts have been inserted in the past. In this group, complications can occur after many years. We report on a 13-year-old girl with pulmonary hypertension caused by chronic thrombo-embolism from a ventriculo-atrial shunt.


Statural growth in Williams-Beuren syndrome. The spontaneous growth of 165 patients (75 girls and 90 boys) with Williams-Beuren syndrome was analysed in a mixed longitudinal and cross-sectional manner. Mean (+/- 1 SD) length at birth was 48.2 +/- 2.6 cm in girls (n = 52) and 49.0 +/- 3.0 cm in boys (n = 65). Intrauterine growth retardation (length below -2 SD of the normal population) was present in 35% of the girls and 22% of the boys. Poor growth was noted during the first 2 years of life. Until age 9 years in girls and 11 years in boys, mean growth followed the 3rd percentile. A pubertal growth spurt with normal growth rate was seen at age 10 years in girls and 13 years in boys, i.e. 1 to 2 years earlier than normal. Menarche also occurred earlier than normal at a mean age of 11.6 +/- 1.5 years (n = 28). Mean adult height was 153.9 +/- 6.9 cm in girls (n = 17) and 168.2 +/- 6.9 cm in boys (n = 27), approximately corresponding to the 3rd percentile in both sexes and correlating with the genetic height potential (target height). The mean deficit of adult height compared to target height was 10.2 cm in girls and 9.1 cm in boys. Skeletal development progressed at an approximately normal rate in both sexes.


Simultaneous measurement, using flow cytometry, of radiosensitivity and defective mitogen response in ataxia telangiectasia and related syndromes. In a retrospective study, peripheral blood mononuclear cells from 13 patients with known ataxia telangiectasia (AT) (Louis Bar syndrome, McKusick #20890) were irradiated with different doses of X-rays prior to stimulation with phytohaemagglutinin. Mitogen response and cell cycle progression were assessed by two-parameter 5-bromo-2'-deoxyuridine/Hoechst--ethidium bromide flow cytometry. Compared to age-matched controls, AT cells show a severely defective mitogen response in both unirradiated and irradiated cells. Following irradiation with 1.5 Gy, AT cells exhibit significantly greater accumulations of cells in the G2 phase of the first cell cycle than controls. The ratio between the number of cells accumulated in the first cycle G2 phase and the growth fraction provides a clear distinction between AT and control cultures. In addition, two patients with microcephaly, normal intelligence, immunodeficiency, chromosomal instability and risk for lymphoreticular malignancies (Seemanová syndrome) and two patients with the Nijmegen breakage syndrome (both syndromes are listed as McKusick #25126) also exhibit very poor mitogen response and moderately increased G2 phase accumulations after X-irradiation. The simultaneous assessment of radiosensitivity and mitogen response in a single cell kinetic assay provides a speedy and accurate classification of cells of AT and AT-related syndromes.


Carrier detection in X-linked adrenoleukodystrophy by determination of very long chain fatty acid levels and by linkage analysis. Diagnosis of X-linked adrenoleukodystrophy is based upon demonstration of high levels of very long chain fatty acids. More recently, in addition to biochemical analysis, closely linked DNA probe St14 has been used for prenatal diagnosis in informative families. Identification of heterozygotes is particularly important, both in order to specifically address only carrier females to prenatal diagnosis, and because appropriate dietary therapy is now available to treat those heterozygotes presenting with neurological symptoms. We report two pedigrees in which carrier detection was performed by a combination of biochemical and molecular genetic analysis. Such approach should allow extremely high accuracy in carrier detection.


Serum levels of carboxyterminal propeptide of type I procollagen in healthy children from 1st year of life to adulthood and in metabolic bone diseases. Type I collagen is the major component of bone matrix; circulating carboxyterminal propeptide of type I procollagen (P-I-CP) levels reflect type I collagen synthesis in tissues and may be an useful index to investigate bone metabolism. We measured P-I-CP by a new radioimmunoassay in 300 healthy children and adolescents and in 40 healthy adults to provide reference data for P-I-CP values. In addition, 79 patients with diagnosed disorders of phospho-calcium metabolism (rickets, vitamin D deficient and vitamin D resistant, hyperparathyroidism, hypo- and pseudo-hypoparathyroidism, osteopenia) were evaluated. In the healthy subjects, serum P-I-CP values were higher in children than in adults; variations of P-I-CP levels were observed according to age and sexual maturation: higher values were found in the first years of life and during pubertal development; pubertal increase reflects the different timing of pubertal development in the two sexes. P-I-CP levels were increased in primary hyperparathyroidism and reduced in diseases related to impaired secretion or action of parathyroid hormone. Higher P-I-CP levels were found in vitamin D deficient and vitamin D resistant rickets. P-I-CP was reduced in anorexia nervosa and during chronic glucocorticoid treatment while it was increased in thyrotoxic osteoporosis. In idiopathic juvenile osteoporosis, P-I-CP values ranged from reduced to increased values. We conclude that P-I-CP may represent an additional biochemical marker of bone metabolism. Since age-related variations are present, reference data for the various ages are need for clinical application of this assay.


High frequency oscillatory ventilation and extracorporeal membrane oxygenation in severe persistent pulmonary hypertension of the newborn. We report on 50 term and near-term neonates (birth weight greater than 1800 g, gestational age greater than 33 weeks) with severe persistent pulmonary hypertension of the newborn (PPHN), referred to us from January 1987 to July 1991 after failure of maximum conventional treatment. All infants had paO2 less than 45 mm Hg when ventilated with peak inspiratory pressure greater than 38 cm H2O and FiO2 = 1.0, hence meeting entry criteria for extracorporeal membrane oxygenation (ECMO). High frequency oscillatory ventilation (HFOV) was tried in all patients. If sufficient oxygenation could not be achieved (paO2 less than 40 mm Hg for at least 2 h), ECMO therapy was begun, which was the case in 25 children. Neonates responding to HFOV (n = 25) were of a slightly younger gestational age (37.0 weeks vs 38.8 weeks, P less than 0.05), had higher Apgar scores and were less hypoxaemic before HFOV (paO2 36.6 mm Hg vs 28.8 mm Hg, P less than 0.01); during HFOV there was a significant rise in paO2 (greater than 150 mm Hg; P less than 0.001) and a fall in pCO2 to 21.6 mm Hg (P less than 0.001). Due to air leaks, which was the main complication of HFOV (52%), ECMO therapy had to be begun in two additional infants after an initial positive effect. HFOV tended to be successful in cases of primary PPHN, meconium aspiration and sepsis, but not in infants with lung hypoplasia as a result of diaphragmatic hernia or other reasons. Success or failure of HFOV could not be reliably predicted by any parameter.(ABSTRACT TRUNCATED AT 250 WORDS)


Long-term low-dose cyclosporin A in steroid dependent nephrotic syndrome of childhood. Therapy of steroid-dependent idiopathic nephrotic syndrome is often unsatisfactory. Since 1986 we have treated nine children (six male and three female), aged 3-16 years, with cyclosporin A (CsA) during 2.0-5.2 (median 3.1) years. All had minimal change disease on renal biopsy and had previously received cyclophosphamide. Mean daily dosage of CsA was 4.1 mg/kg (range 2.7-5.8) and mean whole blood trough level was 220 ng/ml (range 141-271). The relapse rate decreased from 3.4/patient year before CsA to 0.55 on CsA. Discontinuation of CsA or reduction below 2 mg/kg daily was always followed by a relapse. The overall relapse rate, including the period with very low-dose CsA, was 0.95/patient year. Four patients required additional low-dose alternate-day prednisone. Repeat renal biopsy showed minimal change disease in eight patients and focal segmental glomerulosclerosis in one; CsA-toxicity was mild in two and moderate in one. The latter was the only patient with slightly reduced glomerular filtration rate. Two boys with delayed puberty spontaneously matured and reached expected final height. We conclude that long-term low-dose CsA is very effective and steroid-sparing. Its use is justified in selected patients, particularly in those with numerous relapses and in male patients before and during puberty, as long as renal function and CsA-toxicity are carefully monitored.


Cortical visual impairment following bacterial meningitis: magnetic resonance imaging and visual evoked potentials findings in two cases. Cortical visual impairment (CVI) following bacterial meningitis is a very uncommon complication. Two children with CVI following bacterial meningitis are reported. Bacterial agents were Haemophilus influenzae type B in one and meningococci in the other child. Both children showed only insufficient recovery from CVI, mental retardation and residual neurological symptoms. Flash visual evoked potentials (VEP) showed preserved cortical response at onset of CVI. Re-evaluations several months later showed significantly reduced amplitudes, but normal latencies for P100. Thus, flash VEP does not allow prediction of visual outcome. MRI results have not been reported before. MRI at onset of diagnosis showed occipital parenchymal irregularities with enlarged sulci and subarachnoid spaces. Follow up MRI 15 months after onset of CVI in one patient showed marked atrophy of the occipital cortex, hyperintensities of the cortical white matter and no visible optic radiation. The MRI findings indicate hypoxic-ischaemic lesions in the border zone between the distribution of the great cerebral arteries.


Follow up study of cow's milk protein intolerant infants. Over a period of 4 years, 88 infants with cow's milk protein intolerance (CMPI) were followed prospectively in order to evaluate the persistence of CMPI and its relationship between either serum IgE levels or RAST results for cow's milk. After exclusion of lactose intolerance, two positive cow's milk elimination challenge tests were considered diagnostic for CMPI. At the age of 1, 2, 3 and 4 years respectively, 85%, 78%, 49% and 33% of the children still were cow's milk intolerant. Initial serum values of IgE greater than or equal to 10 kU/l indicated a late development of tolerance to cow's milk proteins. At the age of 4 years, 90% of infants with initial IgE levels less than 10 kU/l had become tolerant to cow's milk while this was the case for only 47% of infants with initial IgE levels greater than or equal to 10 kU/l. Initial RAST results for cow's milk bore no obvious relationship to outcome.


Risks of alternative nutrition in infancy: a case report of severe iodine and carnitine deficiency. A 7.5-month-old infant with failure to thrive, developmental delay, muscular hypotonia, a visible goitre and severe osteopenia is described. Laboratory examination revealed a markedly increased serum TSH with low free T4, severe iodine and carnitine deficiency. The infant was breastfed until the age of 2.5 months and was then given a mixture of almond extract in water. The mother is a strict vegan and the father a lactovegetarian. The nutritional intake of the child was severely depleted in calories (-46%), calcium (-73%) and iodine (-88%). The restrictive alternative nutrition was responsible for the various deficiency disorders.


Disposition and metabolism of pravastatin sodium in rats, dogs and monkeys. Pravastatin sodium (pravastatin) is a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and was found to be highly effective in animals and humans, in lowering the plasma cholesterol level by inhibiting cholesterol synthesis selectively in the liver. In the present study the disposition and metabolism of pravastatin was studied in rats, dogs and monkeys using [14C]-labelled compound. The extent of absorption was approximately 70% in rats and 50% in dogs. Tissue distribution examined by both whole-body autoradiography and radioactivity measurement demonstrated that the drug was selectively taken up by the liver, a target organ of the drug, and excreted via bile mainly in unchanged form. Since pravastatin excreted by the bile was reabsorbed, the enterohepatic circulation maintained the presence of unchanged pravastatin in the target organ. The profiles of metabolites were studied in various tissues and excreta and a metabolic pathway of pravastatin was proposed.


In vitro forecasting of drugs that may interfere with codeine bioactivation. The O-demethylation of codeine (methylmorphine) into morphine is mediated by the polymorphic cytochrome P450 DB1 (P450 IID6). By means of in vitro screening in human liver microsomes we have studied the effect on codeine bioactivation of several drugs used as analgesics or as adjuvants for pain control. In microsomes from an extensive metabolizer subject, paracetamol (acetaminophen) and NSAIDs (acetylsalicylic acid, diclofenac, indomethacin, piroxicam, and pirprofen), benzodiazepines (chlordiazepoxide, clonazepam, diazepam, flunitrazepam, and midazolam), and anticonvulsants (carbamazepine and phenytoin) did not alter the reaction. There was marked inhibition of in vitro morphine production by neuroleptics (chlorpromazine, haloperidol, levomepromazine, and thioridazine), metoclopramide, and tricyclic antidepressants (amitriptyline, clomipramine, desipramine, imipramine, and nortriptyline). Enzyme kinetics showed competitive inhibition by neuroleptics (chlorpromazine Ki = 0.5 microM) and antidepressants (clomipramine Ki = 6.8 microM), which are substrates of the polymorphic monooxygenase. Due to the low affinity of codeine for P450 DB1 (Km = 100-200 microM), its bioactivation in extensive metabolizers, and thus its analgesic efficacy, is liable to vary greatly when it is combined with any drug that has a high affinity for the polymorphic isozyme.


Bioconversion of netobimin pro-drug by gastrointestinal fluids of ruminants. The biotransformation of netobimin (NTB) pro-drug by ruminal, abomasal and intestinal fluids of sheep and cattle was investigated under anaerobic conditions in vitro. No metabolic conversion of NTB was observed upon incubation with abomasal fluid or boiled samples of different gastrointestinal fluids. NTB pro-drug was reduced and cyclised into albendazole (ABZ) and this further oxidized into ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) by both sheep and cattle ruminal and ileal fluids. A zwitterion formulation of NTB produced a significantly greater amount of anthelminthically active ABZ metabolites (ABZ and ABZSO) than a trisamine salt formulation of the same compound. Although there was no difference in the total amount of products formed, both cattle ruminal and ileal fluids showed a greater oxidative capacity than sheep derived fluids. This was evidenced by the greater amounts of ABZSO recovered and by the resultant lower ratios of ABZ/ABZSO obtained with cattle fluids.