New noncorneal HK-loop electrode for clinical electroretinography.

A new noncorneal electrode for clinical electroretinography was developed. It consists of a thin wire forming a loop modeled to fit into the lower conjunctival sac. Electrical contact is made with the scleral conjunctiva through an exposed portion of otherwise insulated wire. The recorded pattern electroretinograms are in the same amplitude range as if recorded by the gold foil electrode, while the flash electroretinograms with the new electrode are of about two-thirds the amplitude of corneal electrodes. The new electrode is more durable and hence less expensive than gold foil electrodes and can likewise be used without topical anesthetic. Cleaning is easy and effective. The electrode rarely causes discomfort and produces stable responses for at least 2 hours. The electrode aims to match stability of skin electrodes with sensitivity of fragile foil and fiber electrodes.


Long chain polyunsaturated fatty acids and fetal growth.

Long chain polyunsaturated fatty acid composition of plasma choline phosphoglycerides has been measured at birth in 22 preterm infants. Positive correlations were found between both n-6 and n-3 fatty acids and measurements of growth and maturation. 20:4(n-6) and the sum of 20:3(n-6) + 20:4(n-6) correlated most strongly with weight and head circumference, while 22:6(n-3) showed strongest correlation with length of gestation. These findings are of relevance to understanding the role of nutrition in fetal growth and in establishing the group of infants most at risk of postnatal deficiency of essential fatty acids.


The relationship between the quantity and quality of prenatal movements in pregnancies complicated by intra-uterine growth retardation and premature rupture of the membranes.

In 17 fetuses with intrauterine growth retardation (IUGR), we studied the quantity of general movements and fetal breathing movements both cross-sectionally and longitudinally. In IUGR fetuses, cross-sectional comparisons were made between the quantity of fetal movements and (1) the fetal clinical condition and (2) the quality of general movements. In addition, the quantity of fetal movements in IUGR was compared with that in uncomplicated pregnancies and in pregnancies complicated by premature rupture of the amniotic membranes. In IUGR, the quantity of general movements declined from 25 weeks gestation onwards, whereas the quantity of fetal breathing movements increased. Longitudinal assessment of these parameters was obtained in four cases and showed a decline of general movements. No relationship between prenatal longitudinal data and neonatal outcome could be observed. Relating the quantity of general movements and breathing movements to the fetal condition, growth retarded fetuses were divided into three groups according to fetal deterioration. 1. Normal amount of amniotic fluid and normal fetal heart rate patterns. 2. Reduced amount of amniotic fluid. 3. Abnormal fetal heart rate patterns. The quantity of general movements as well as that of breathing movements was low in group 3, compared to group 1. In group 2 only the quantity of breathing movements and not of general movements was low. A similar pattern was found in the relation with the quality of general movements observed during fetal deterioration. Cross-sectional analysis of median values (28-31 weeks gestation) did not reveal differences in the quantity of general movements when IUGR, normal pregnancies and premature rupture of the membranes (with or without oligohydramnios) were compared. The quantity of fetal breathing movements was significantly lower in pregnancies complicated by IUGR and by premature rupture of the membranes with oligohydramnios compared to those of normal pregnancies and premature rupture of the membranes without oligohydramnios. In uncomplicated IUGR, the quantity of general movements and breathing movements is in the same range as in normal uncomplicated pregnancies. Similar to the quality of general movements, the quantitative variables were related to the fetal condition. However, in contrast to the quality of general movements, the quantity of general movements and breathing movements showed a high inter- and intraindividual variation. Therefore, the results of this study discourage the use of quantitative aspects of general movements and breathing movements as reliable indicators of the neurological condition in the individual fetus.


The influence of blood transfusion on brain blood flow autoregulation among stable preterm infants.

Prior to and 24 h following blood transfusion serial determinations of both cerebral artery flow velocity waveforms and mean arterial blood pressure have been used to reconstruct the autoregulatory curve and its upper blood pressure limit among five stable preterm infants. Prior to transfusion the autoregulatory range of cerebral blood flow (CBF) was narrow due to a relatively low-set upper blood pressure limit. At 24 h after transfusion each individual has been re-examined. Following correction of anemia both a significant reduction of CBF velocities as well as a concomitant rise of the Pulsatility Index (PI) occurred over the entire range of blood pressures indicating a reduction of CBF after transfusion. In addition a right-sided shift of the upper limit towards higher mean blood pressures occurred after transfusion and resulted in an extension of the autoregulatory plateau of CBF. These favourable effects of blood transfusion ameliorating autoregulation of brain blood flow particularly at higher blood pressures might well bear important therapeutic perspectives in our effort to prevent intracranial haemorrhage among sick preterm infants.


Normal auditory brain stem evoked responses in infants of diabetic mothers.

Auditory brain stem responses potentials were recorded from 71 newborns within the first 2 weeks after birth; conceptional age ranged from 37 to 41 weeks. Thirty-nine newborns were infants of diabetic mothers (IDMs) (17 White A, 22 White B-D) and 32 healthy term newborns served as control group. IDMs with additional high risk for cochlear or brain stem integrity were excluded. Birthweight for gestational age was significantly higher for IDMs. No differences in auditory brain stem responses wave latencies or amplitudes were observed between healthy infants of the control group and IDMs.


Effect of morphine and pancuronium on the stress response in ventilated preterm infants.

Ninety-five premature newborns who had hyaline membrane disease and were struggling against the ventilator were randomised to one of three treatment groups: morphine (group M), pancuronium (group P) or morphine with pancuronium (group M+P). The dose of morphine was 50 micrograms/kg per h but was increased to 100 micrograms/kg per h in group M infants if they continued to struggle. The dosage of pancuronium was 100 micrograms/kg given as required to inhibit spontaneous respiration. Plasma catecholamine levels were measured on entry and at 24 h. Blood pressure and ventilatory requirements were determined on entry and at 6 h. The clinical outcome of the infants was documented. Group M infants (n = 29) showed a significant reduction in noradrenaline levels (median change -2.2 nmols/l (range -47.2 to +7.2 nmols/l), although seven were withdrawn from this group because of failure to settle. Group P (n = 28) and group M+P (n = 38) showed no significant change in noradrenaline levels. Comparison between the groups showed that group M infants had a significant reduction in noradrenaline levels compared with group P. The immediate effects of treatment on blood pressure and ventilatory requirements were similar in the three groups. The clinical outcome did not differ for any of the measured parameters. When adequate sedation is achieved, morphine may reduce the stress of newborn intensive care.


Abnormal internal carotid and umbilical artery Doppler in the small for gestational age fetus predicts an adverse outcome.

Doppler studies of the internal carotid and umbilical arteries were performed in 29 women admitted to the antenatal ward with a diagnosis of being small for gestational age. The primary aim of the study was to determine whether an abnormal internal carotid waveform was associated with a poor perinatal outcome defined as: fetal distress, acidosis at birth or perinatal death. A poor perinatal outcome occurred in 18 pregnancies including six perinatal deaths. There was a highly significant association between an abnormal internal carotid waveform and a poor outcome and this was particularly so at a gestational age of < or = 34 weeks where the sensitivity, specificity and predictive values were all 100%. No baby with a normal internal carotid waveform had a poor outcome. Abnormal umbilical waveforms tended to occur earlier in pregnancy than abnormal internal carotid waveforms and were also predictive of an adverse perinatal outcome. Whether this information should be used clinically is not clear as abnormal umbilical artery and internal carotid waveforms were sometimes present for weeks before delivery occurred for fetal reasons.


Prospective study of laparoscopic cholecystectomy in two hundred and fifty patients.

Two hundred and fifty consecutive patients (151 women, 99 men, age 49.3 years) with symptomatic gallstones as diagnosed on ultrasonography underwent elective laparoscopic cholecystectomy over a 12-month period. Preoperative intravenous cholangiography (IVC) and endoscopic retrograde cholangiography (ERC) were also performed in 203 and 200 patients, respectively. Laparoscopic cholecystectomy was successfully performed on 248 of the 250 patients (99.2%). Two patients required conversion to open cholecystectomy because of a pin hole injury to the portal vein in one case, and because of technical difficulties with the dissection due to severe adhesions in the other patient. Procedure time in the initial 100 cases decreased from an average of 216 minutes for the first ten cases to 87 minutes for the last 30 cases, including the time required for intraoperative cholangiography (IOC). Bile duct stones were found in 4 cases on IOC which were diagnosed by preoperative ultrasonography, IVC and ERC in 0, 2 and 3 cases respectively. An intraabdominal drain was inserted for two days in all cases. The mean hospital stay was 8.6 days (range 4 to 19 days) with no readmissions. No complications were seen at short-term follow-up one month after discharge. Although there was a significant learning period, the procedure was safe and effective and could be performed with minimal risk. The results show that physicians with experience in both endoscopy and laparoscopy are well qualified to perform laparoscopic cholecystectomy after appropriate training.


Prospective evaluation of the use of endoscopic retrograde cholangiography prior to laparoscopic cholecystectomy.

Preoperative cholangiography and subsequent removal of bile duct stones may increase the efficacy of laparoscopic cholecystectomy and reduce the rate of conversion to open cholecystectomy. Since there is little data on the incidence of choledocholithiasis in this group of patients, we undertook a prospective study on the routine performance of ERC in 288 patients selected for laparoscopic cholecystectomy. ERC succeeded in 264 of the 288 patients (91.7%) and showed a normal bile duct system in 227 (86.0%). Atypical bile duct anatomy was seen in eight patients. Open cholecystectomy was performed in seven of them but was judged to be absolutely necessary in only two cases (one patient each with Caroli syndrome and Mirizzi syndrome). ERC also revealed bile duct stones in 29 of 264 patients (11.0%) which had not been suspected on the basis of clinical, laboratory and ultrasonographic findings in nine cases (3.4%). EPT succeeded in all of the 29 patients with choledocholithiasis but open cholecystectomy was subsequently performed in four patients due to incomplete bile duct clearance (n = 3) or temporary bleeding after EPT (n = 1). The rate of ERC/EPT-related morbidity was 2.8%. It is concluded from a risk-benefit analysis in these patients that ERC should be restricted to patients with suspected bile duct stones. Following this strategy, small ductal concrements and bile duct abnormalities will be missed in 6.4% of cases but the clinical relevance of these findings is still unclear. In patients with combined gallbladder and common bile duct stones, preoperative EPT plus subsequent laparoscopic cholecystectomy appears to be an effective and time-saving therapeutic regimen which should be compared with open cholecystectomy plus common bile duct exploration in future studies.


Preliminary experience using laparoscopic transcystic cholangioscopy for treatment of common bile duct stones.

Laparoscopic transcystic cholangioscopy (LTC) in combination with electrohydraulic lithotripsy may be an alternative treatment to ERCP and sphincterotomy in patients with both gallbladder and common bile duct stones undergoing laparoscopic cholecystectomy. Preliminary experience using LTC lithotripsy in 13 cases is reported. In 12 cases the stones were pushed out into the duodenum using the tip of the cholangioscope, in 8 of them stone disintegration via LTC lithotripsy also being required. In the remaining case the cholangioscope could not be inserted into the common bile duct via the cystic duct due to complete cystic duct obstruction. The average hospital stay was 9 days (range 6-16) in patients with LTC/lithotripsy, which did not differ significantly from 8.4 days (range, 4-19) (n = 330) in the group undergoing laparoscopic cholecystectomy alone. The patients usually resumed normal activity the day after discharge. LTC lithotripsy has the advantages over endoscopic sphincterotomy of a shorter treatment and preservation of a normal functioning sphincter of Oddi. Further technical improvements, especially the development of a cholangioscope for this purpose, are urgently required.


Long-term follow-up after laparoscopic cholecystectomy.

A five-year follow-up was performed in 93 of 94 patients who successfully underwent laparoscopic cholecystectomy between 1985 and 1987. The results were compared to those in 130 of 136 patients of a non-randomized control group who had undergone open cholecystectomy during the same period. Patients characteristics and gallbladder findings were comparable in the two groups. Postoperative complications were infrequent and not significantly different in the two groups, whereas the mean hospital stay was shorter in the laparoscopic group. During follow-up, one patient in the laparoscopically treated group had a common bile duct stone which was treated surgically since he refused to undergo ERCP; this patient died of postoperative complications. Various abdominal complaints were reported in 27% and 25% of the patients undergoing laparoscopic and open cholecystectomy, respectively; in most cases no organic causes were found. Scar problems were less frequently reported in the laparoscopically treated group (2% versus 12%). It is concluded that the long-term results of laparoscopic cholecystectomy are as good as those of open cholecystectomy, however, the hospital stay is significantly shorter and the long-term cosmetic results are considerably better in laparoscopically treated patients.


In vitro fragmentation of gallstones with lithotriptors for use in laparoscopic cholecystotomy.

In preliminary in vitro experiments, five different lithotriptors for use in laparoscopic cholecystotomy were tested to determine the fragmentation time and rate of a defined group of gallstones. The Alexandrit laser and the ultrasonic lithotriptor were too ineffective to warrant further investigation. In phantom tests with pig gallbladders, the pulsed dye laser and the electrohydraulic lithotriptor had a fragmentation rate of 100%, but the rate of clearing of the gallbladder was 0% as stone debris always remained in the gallbladder. The clearing rate of the RotoLith mechanical lithotriptor was 84.6%. The RotoLith lithotriptor was the most effective device because the stones were ground into such small particles that they could easily be rinsed out of the gallbladder. As the RotoLith procedure does not need to be monitored optically, the opening of the gallbladder need not exceed 3 mm compared to 10 mm for the other lithotriptors. This makes closure of the gallbladder safer and it would seem feasible to perform the procedure using the RothoLith lithotriptor under local anaesthesia.


Evaluation of four commercial automatic endoscope washing machines.

Four commercially available endoscope washing machines (Pyser System 83, Wolf Fiberscope Disinfector, Olympus EW20, Keymed Auto-Disinfector) were evaluated. In most cases the machines were expensive, immobile and noisy, and could only wash one endoscope per cycle. Daily use involved repetitive chores and contact with glutaraldehyde. Disinfection procedures were flawed; no machine auto-disinfected adequately. In conclusion, these machines are unsatisfactory and represent a potential source of serious infection.


Hemodynamic changes during laparoscopic cholecystectomy in patients with heart disease.

Changes in the hemodynamics and urine output were investigated in 19 patients undergoing laparoscopic cholecystectomy, five of whom had heart disease with the New York Heart Association classification I (n = 1) and II (n = 4). Systemic blood pressure, central venous pressure, pulmonary capillary wedge pressure and cardiac output did not significantly change during the procedure including the establishment of pneumoperitoneum. Urine output 30-60 min after starting the pneumoperitoneum was significantly lower in the patients with heart disease compared to the values before and in the initial phase (0-30 min), and also to the values before and during the procedure in the control group. One patient suffered temporary cardiac decompensation following laparoscopic cholecystectomy which prolonged his hospital stay to seven days. The remaining four patients with heart disease could be discharged on the third or fourth day postoperatively. It is concluded that laparoscopic cholecystectomy is feasible in patients with heart disease but attention should be paid to the possibility of oliguria during prolonged pneumoperitoneum.


Preliminary experience using endoscopic ultrasonography in the diagnosis of choledocholithiasis.

Twenty patients with symptomatic cholelithiasis and suspected choledocholithiasis were evaluated in an ongoing prospective trial using endoscopic ultrasonography (EUS), standard abdominal ultrasonography (US) and ERCP for the detection of choledocholithiasis prior to laparoscopic cholecystectomy. EUS was used successfully to image the extrahepatic bile duct in all patients. EUS detected three of four proven bile duct stones and correctly identified 16 bile ducts as stone free, thus being more accurate than standard abdominal US. The preliminary results of this ongoing prospective trial and the experience reported by other authors suggest that EUS may be as sensitive as ERCP in the detection of choledocholithiasis.


Duodenal perforation after laparoscopic cholecystectomy.

A case is reported of duodenal perforation complicating laparoscopic cholecystectomy performed by laser dissection. The importance of investigating a patient with persistent shoulder-tip pain following this technique to exclude a subphrenic abscess is emphasised.


Successful treatment of an impacted common bile duct stone with intraoperative choledochoscopic electrohydraulic lithotripsy.

An impacted distal common bile duct stone which was encountered during supraduodenal common bile duct exploration could not be retrieved using stone forceps, a basket, normal saline flushing or a Fogarty catheter. Using intraoperative choledochoscopic electrohydraulic lithotripsy the stone was fragmented and successfully removed with a basket and normal saline flushing. The patient was well after a follow-up period of 18 months.


Endoscopic management of cystic duct fistulas after laparoscopic cholecystectomy.

Two patients are reported who developed bile ascites as a result of cystic duct fistulas following laparoscopic cholecystectomy. Both patients were successfully treated with endoscopic retrograde cholangiopancreatography, utilizing sphincterotomy and nasobiliary tube placement. Characteristics of this syndrome and advantages of this form of therapy are emphasized.


Laparoscopic cholecystectomy in a patient with empyema of the gallbladder and situs inversus.

The successful performance of laparoscopic cholecystectomy in a patient with situs inversus viscerum and empyema of the gallbladder is reported. The 62-year-old man presented with pain in the left upper quadrant associated with fever, chills, nausea and vomiting. The abdomen was tender with guarding and a palpable globular mass in the same region. CT scan demonstrated a distended gallbladder with thick shaggy walls which contained a 2 cm gallstone in the neck and also revealed dextrocardia and situs inversus. The patient's postoperative recovery was uneventful.


Mouse hypersensitivity pneumonitis: depletion of NK cells abrogates the spontaneous regression phase and leads to massive fibrosis.

The contribution of natural killer cells (NK) in the progression of mouse hypersensitivity pneumonitis induced by repeated intranasal instillations with the thermophilic actinomycete Faeni rectivirgula was examined. These instillations determined a very large increase in the lung index (ca. twofold at 3 weeks), used as a measure of inflammation. In addition, this instillation was associated with a tenfold increase in the number of cells recovered in the bronchoalveolar lavage (BAL) at 3 weeks and thereafter. Most of these cells were macrophages, whereas T lymphocyte numbers increased at 6 weeks and thereafter. The instillations were also associated with a substantial fibrotic response in the lungs, as seen by large increases in hydroxyproline levels in the lungs. This fibrotic response, however, diminished after 6 weeks of instillations. Similarly, examination of histological preparations of lungs of challenged mice showed that F. rectivirgula induced inflammatory infiltrates of macrophages, lymphocytes, and neutrophils. The severity of the lesions were reduced in mice given more than 6 weeks of the actinomycete challenge. The involvement of NK cells on the development of this pulmonary pathology was determined by infusing F. rectivirgula-challenged mice with anti-NK 1.1 antibody. The depletion protocol was validated by verifying that such treatments effectively blocked lung NK cell activity. Such NK cell-depleted mice responded to the F. rectivirgula challenge with an increased lung index at 9 and 12 weeks, compared to mice challenged with F. rectivirgula and given control antibody. NK cell-depleted mice also responded to the actinomycete with a superior cellular recruitment in the BAL, with this increase mostly mediated by macrophages. Similarly, NK cell-depleted mice developed a fibrotic response that was much higher than that seen in control challenged mice, at 6, 9, and 12 weeks after initiation of the transnasal instillations. This was corroborated by scoring the severity of the histopathological lesions, which showed that NK cell-depleted mice had more severe lesions than challenged control mice. The importance of NK cells was confirmed by demonstrating that mice given anti-NK 1.1, challenged with F. rectivirgula and reconstituted with Percoll gradient-enriched lung NK cells had hydroxyproline levels at 9 and 12 weeks that were comparable to that seen in intact mice, as well as a histopathological score similar to control challenged mice. Overall, this suggests that in the course of a pulmonary inflammatory response, NK cells exert a suppressive effect on cellular recruitment in the BAL, contribute to down-regulating the inflammatory response, and are involved in blocking the appearance of fibrosis.


Developmental regulation of surfactant-associated proteins in rabbit fetal lung in vivo.

The developmental regulation of the rabbit surfactant-associated proteins, SP-A, SP-B, and SP-C, was investigated using Northern blot analysis. These proteins comprise approximately 10% by weight of pulmonary surfactant, a lipoprotein secreted by type II cells that reduces surface tension at the air-alveolar interface. SP-A mRNA and SP-B mRNA were first detected in rabbit fetal lung at day 24 of gestation (term = 31 days), i.e., approximately two days prior to the appearance of lamellar bodies within differentiated alveolar type II cells. The relative abundance of SP-B mRNA detected on day 24 of gestation was greater than that of SP-A mRNA. Fetal lung SP-A mRNA and SP-B mRNA levels increased rapidly during the remainder of gestation, reaching a maximum at day 31 of gestation. The relative concentrations of SP-A mRNA and SP-B mRNA were decreased in day 2 neonatal and adult lung tissues when compared to the levels present in fetal lung tissue late in gestation. A 0.5-kb rabbit SP-C cDNA was generated using the reverse transcriptase-polymerase chain reaction and was found to have high sequence homology to the human and rat SP-C cDNA nucleotide sequences. The predicted amino acid sequence for the rabbit SP-C cDNA revealed strong conservation of a hydrophobic region close to the amino terminus of the SP-C protein. Fetal lung SP-C mRNA was detected at day 19 of gestation, the earliest time point examined in this study. SP-C mRNA levels gradually increased in fetal lung tissue until day 28 of gestation and then remained level throughout the remainder of gestation and in the day 2 neonatal and adult rabbit lung tissue. These results suggest that the developmental pattern of induction of mRNA for the surfactant-associated proteins, SP-A, SP-B, and SP-C, differ from each other and are different in several respects from the developmental patterns observed in fetal lung tissue of the rat and human species.


Results of a multicentre European survey in 1991 of metronidazole resistance in Helicobacter pylori. European Study Group on Antibiotic Susceptibility of Helicobacter pylori.

In 1991 a multicentre survey on the prevalence of metronidazole resistance in Helicobacter pylori in vitro was carried out in 12 hospitals in 11 different European countries. The susceptibility of Helicobacter pylori to metronidazole was determined in each centre by measuring the MIC on agar with the PDM E-test (AB Biodisk) according to a standard procedure. Overall, 122 of 443 (27.5%) strains tested were resistant to metronidazole (MIC > 8 micrograms/ml). The level of resistance to metronidazole varied markedly between centres (from 7% to 49%) and was found to be substantially higher in Africans and other non-Caucasian subjects than in natives from European countries. The overall rate of resistance to metronidazole was higher in females (34.7%) than in males (23.9%), and varied according to the age group, being highest among women aged 20 to 39 (50%). Previous use of metronidazole was reported in only 16 patients, 11 of whom (68.8%) harboured resistant Helicobacter pylori strains. Although differences in the rate of metronidazole resistance in Helicobacter pylori most probably relate to variations in use of this drug in different populations, such use may frequently go unrecognized. This study emphasises the importance of monitoring the drug resistance of Helicobacter pylori on a local basis. Standardisation of the methods for testing the susceptibility of Helicobacter pylori in vitro is clearly needed for this purpose.


Inhibition of neutrophil adherence and movement by acute cigarette smoke exposure.

Peripheral blood neutrophils were harvested and exposed acutely in vitro to physiologically attainable levels of cigarette smoke. The adherence of radiolabeled neutrophils subsequently to alveolar epithelial cell monolayers was measured. In contrast to control cells, smoke-exposed neutrophils were significantly less adherent and failed to increase their adherence following stimulation with phorbol ester or f-met-leu-phe (fMLP). Flow cytometric analysis of the cell surface adhesion protein CD18 demonstrated no significant change in expression following in vitro smoke exposure and, furthermore, no increase in surface CD18 of smoke-exposed cells following consecutive fMLP stimulation was demonstrated. Acute in vivo cigarette smoking of up to 4 cigarettes also did not alter peripheral blood neutrophil CD18 expression. Cell spreading and chemokinesis, but not chemotaxis, was also impaired following in vitro smoke exposure. These data suggest that acute cigarette smoke may impair the crucial neutrophil functions of adherence and movement. However, the chronic effects of cigarette smoke exposure may clearly differ.


Patterns of multiple resistance to antibiotics in gram-negative bacteria demonstrated by factor analysis.

Principal component analysis was used to demonstrate the main associations between patterns of resistance to antibiotic drugs in 670 gram-negative bacteria consecutively isolated from blood cultures over a period of two years. Six factors were derived, which accounted for 84% of the total variance of the original matrix. Each factor represented an association between resistance to certain antibiotics as follows: factor 1: aztreonam, third generation cephalosporins and aminoglycosides; factor 2: first and second generation cephalosporins; factor 3: tetracycline and chloramphenicol; factor 4: ampicillin and ureidopenicillins; factor 5: trimethoprim/sulfamethoxazole; factor 6: fluoroquinolones. On two-way analysis of variance the difference in the factor scores was significant between bacteria for all factors except factor 5. The difference in factor scores between community and hospital acquired strains was significant only for factors 1, 2 and 6. Only the score of factor 6 showed a clear trend to increase with time during the two-year study period. Patients who were treated with antibiotics prior to bacteremia had higher scores for all factors, the difference being most marked in patients treated with fluoroquinolones. Factor analysis can be used to describe phenotypic associations between resistance to antibiotics, and the factor score used to compare groups of isolates and to demonstrate temporal and other trends.


Alpha-naphthyl acetate esterase-1 (ANAE-1) secreted by epithelioid cells from induced rabbit lung granuloma showed MIF activity.

The function of alpha-naphthyl acetate esterase-1, whose isoelectric point values range from 5.15 to 5.45, was examined. A higher value of alpha-naphthyl acetate esterase-1 was detected in the extracts of epithelioid cells isolated from rabbit lung granuloma at 4 weeks after injection of Freund's complete adjuvant, compared to those values of alveolar macrophages isolated from the same lungs described above and of the normal lungs. Additionally, this enzyme activity was observed to be prominent in the culture supernatants of epithelioid cells. alpha-Naphthyl acetate esterase-1 was purified from lung granuloma as a single 62-kDa band by SDS-PAGE analysis. The purified enzyme showed a macrophage migration inhibition activity at concentrations over 20 nM, and its activity was dose-dependent. Moreover, when various amounts of the purified enzyme were added to lymphocyte-derived macrophage migration inhibitory factor, macrophage migration inhibition was significantly enhanced with a dose-dependent manner. The results suggest that alpha-naphthyl acetate esterase-1 secreted by granuloma macrophages, particularly by epithelioid cells, contributes to granuloma formation.


In vitro effects of hyperoxia on alveolar type II pneumocytes: inhibition of glutathione synthesis increases hyperoxic cell injury.

An in vitro model of alveolar epithelial oxidant injury was developed based on exposure to hyperoxia of cultured guinea pig type II pneumocytes using a biphasic cell culture system in aerobiosis. The present study investigates the roles of intracellular antioxidant enzymes and of glutathione in providing protection against hyperoxia. A 2-day type II cell culture in normoxia was associated with a significant decrease in protein, catalase, and Cu-Zn SOD cell content, whereas ATP cell content, Mn-SOD, and glutathione peroxidase (GPx) activities did not change and glutathione cell content significantly increased. Exposure of type II cells to hyperoxia did not induce significant changes in cell content in protein, SOD, catalase, GPx, or glutathione cell content when compared to control cells (exposed to normoxia). With ATP cell content expressed as a cell injury index (CII), type II cell injury was found to increase with increasing O2 concentrations. Indeed, a 2-day 50% O2 and 95% O2 exposure resulted in a CII of -7.5 +/- 6.2% and 17.9 +/- 5.9%, respectively, LDH release by type II cells was not significantly increased after hypoxic exposure. Cell injury effects of hyperoxia did not correlate with the endogenous antioxidant enzyme activities (SOD, Mn-SOD, catalase). In marked contrast, there was a significant correlation between the CII and total glutathione content of type II cells (p < .01). This correlation was largely due to the close relationship between CII and reduced glutathione. Hyperoxic induced cell injury (as demonstrated by CII > 0) was clearly associated with significantly lower intracellular glutathione level when compared to experiments without hyperoxia induced cell injury (CII < 0). In addition, in the presence of buthionine sulfoximine (BSO), the ability of type II cells to synthetize new glutathione was severely impaired, whereas ATP cell content and cell antioxidant enzyme activities did not change. As a consequence, the reduction of intracellular glutathione significantly increased the susceptibility of cells to hyperoxia injury (p < .05). The results strongly support the hypothesis that the regulation of glutathione levels is an important mechanism in protecting hyperoxia-induced type II cell injury.


Scanning electron microscopy of bacterial biofilms on indwelling bladder catheters.

Fifty Foley bladder catheters that had been indwelling for periods ranging from 3 to 83 days (mean 35 days) were examined for the presence of bacterial biofilm. Scanning electron microscopy on freeze-dried cross-sections and fixed, critical point-dried longitudinal sections revealed biofilm formation on the luminal surfaces of 44 of the catheters. Culture of urine samples and sonicates from catheters revealed that the prevalence of bacteriuria was less than that of catheter colonization. A wide range of nosocomial species were found colonizing the catheters, Escherichia coli being most often isolated. The bacterial composition of the biofilms ranged from single species to mixed communities containing up to four species. There was no relationship between the length of time that the catheter had been in situ and the extent of biofilm formation. The biofilms varied in thickness from 3 to 490 microns and were visible as layers of bacterial cells up to about 400 cells deep, embedded in a matrix.


Complex cytokine modulation of a continuous line of mink lung epithelial cells (Mv1Lu).

The continuous mink lung epithelial cell line Mv1Lu has proven to be a sensitive reporter line in the bioassay for purified TGF-beta, exhibiting a sigmoid-shaped concentration-response relationship with an EC50 of 12 pM (0.3 ng/mL). Maximal inhibition of Mv1Lu cells generates a 75-95% decrement in the number of adherent cells. However, this bioassay is not specific for TGF-beta as originally claimed. Mv1Lu cells are sensitive to other cytokines and substances found in complex biological fluids. In this study the effects of other biological response modifiers in this assay were tested and several were found to have important growth modulatory capacities that confound the quantitation of TGF-beta. EGF, TGF-alpha, fibronectin, and IGF-I all induce Mv1Lu cell proliferation. In contrast, neither PDGF (-AA, -AB, -BB) nor endotoxin (< or = 10 micrograms/mL) affect Mv1Lu cell number. TGF-beta and TNF-alpha at high concentrations (> or = 10 ng/mL) are the only cytokines examined that inhibit Mv1Lu proliferation. TGF-beta decreases final cell number both by preventing mitosis and by inhibition of adherence of cells to the uncoated dish. Several strategies are suggested to assure the specificity of this otherwise convenient bioassay for TGF-beta.


Rapid detection of tuberculous and non-tuberculous mycobacteria by polymerase chain reaction amplification of a 162 bp DNA fragment from antigen 85.

A polymerase chain reaction (PCR) assay was developed for detection of mycobacteria using amplification of a 162 bp region of the genes coding for the mycobacterial antigen 85 complex. Strains belonging to the Mycobacterium tuberculosis complex were further differentiated from non-tuberculous mycobacteria by hybridization of the PCR derived Southern blot with an internal oligonucleotide probe and washing under stringent conditions. The method allowed rapid and sensitive detection of mycobacterial DNA in uncultured clinical samples. PCR results obtained for Mycobacterium tuberculosis in 206 specimens from 180 untreated patients gave a sensitivity of 93.9% and a specificity of 94.3% compared with the culture. PCR detected DNA from Mycobacterium tuberculosis in seven samples from patients with clinically evident tuberculosis in whom culture was negative. The results suggest that this PCR assay could be used for early and specific diagnosis of tuberculosis.


Use of genomic fingerprinting in the characterization of Neisseria gonorrhoeae isolated in Valencia, Spain.

Ninety-five Neisseria gonorrhoeae organisms isolated in Valencia, Spain, were characterized by antibiotic sensitivity testing, auxotyping, serotyping, plasmid analysis and restriction endonuclease fingerprinting (HindIII digestion). Cluster analysis of the restriction patterns revealed that 31 isolates (32.6%) formed 12 clearly defined clusters. Penicillinase-producing Neisseria gonorrhoeae strains formed four of these groups. Eight groups of gonococcal strains were identified by auxotyping, although 83% of isolates belonged to two auxotypes (Proto, Pro-). Twenty-three different serovars were identified by serotyping. The serovar pattern IB/rop was found in 38% of isolates. A 60% coincidence was found between gonococcal groupings obtained by combination of auxotyping, serotyping and plasmid analysis and those obtained with the restriction enzyme fingerprinting technique. The specificity of enzyme restriction patterns of Neisseria gonorrhoeae is confirmed to be of practical importance in the epidemiologic study of gonorrhoea.


Technique for calculation of the true costs of antibiotic therapy.

An in-house method for costing antibiotic therapy is presented which quantifies hidden costs including costs arising from intravenous administration, labour, serum antibiotic assay, monitoring of haematological and biochemical indices and disposal of sharp instruments. A study of various hospital procedures relating directly to antibiotic therapy was undertaken in an acute medical ward, which involved determination of staff members performing various procedures, consumables used and time taken. Results of this study facilitated accurate quantification of hidden costs of i.v. antibiotic therapy in this ward. Using these results, the cost of five-day courses of gentamicin, cefuroxime, penicillin G, flucloxacillin and erythromycin were calculated. The costing of adverse effects was not attempted. It is recommended that a costing technique of this sort is used in cost-benefit analysis of antibiotic use, as the cost of the drug alone is misleading.


Penetration of pefloxacin into maxillary sinus cavity and nasal secretions.

The ability of oral pefloxacin to penetrate into maxillary sinus cavity and nasal secretions was studied in 39 patients suffering from an exacerbation of chronic maxillary sinusitis. The mean levels of pefloxacin in sinus aspirate fluid 0, 3, 6, 9 and 12 h after the second oral dose of 400 mg were 2.30, 6.92, 3.74, 3.47 and 2.82 mg/l respectively. In sinus cystic fluid (macroscopically non-purulent) the mean pefloxacin levels were 2.30, 7.15, 4.20 and 4.15 mg/l respectively, and in nasal secretion 1.90, 9.05, 3.71, 3.20 and 2.85 mg/l respectively. The mean pefloxacin levels in serum obtained simultaneously were 1.50, 5.00, 3.10, 2.70 and 2.20 mg/l respectively. It is concluded that pefloxacin accumulates in inflamed sinus fluid at concentrations exceeding blood levels.


Infective endocarditis with involvement of the tricuspid valve due to Capnocytophaga canimorsus.

A case of endocarditis with vegetations on the tricuspid valve caused by Capnocytophaga canimorsus is described. Extensive diagnostic investigations preceded the diagnosis, including blood cultures, 34 of which were sterile. A possible role of the pulmonary circulation in the negative blood cultures is discussed.


Reactivation of Q fever following cardiac surgery.

To determine whether reactivation of Q fever occurs following cardiac surgery 43 patients who had antibodies to Coxiella burnetii were identified pre-operatively. Serum samples were collected 1, 4 to 6, and 24 weeks post-operatively. One patient had active Q fever endocarditis. Seven of the remaining 42 (17%) had a > or = fourfold rise in titres for antibodies to Coxiella burnetii post-operatively. Only two of these seven had an increase in antibody titres for other agents suggesting that the fourfold titre rise for Coxiella burnetii was not part of a polyclonal response. It is concluded that self-limited reactivation of infection may occur among Coxiella burnetii seropositive patients undergoing open heart surgery.


First reported case of Streptococcus pyogenes infection with toxic shock-like syndrome in Italy.

A 43-year-old male who sustained a superficial hand injury developed streptococcal toxic shock-like syndrome and died within 48 hours. The clinical course of the illness in this previously well patient was rapid and fulminant. The organism responsible was a group A beta-hemolytic streptococcus which was identified as opacity factor negative, M serotype 1, T type 1. The organism produced streptococcal pyrogenic exotoxins B and C, but no detectable exotoxin A although it carried speA, the gene for exotoxin A. This is the first case reported in Italy, and further emphasizes the virulence of these organisms and the rapidity with which the illness can progress.


Familial outbreak of agricultural anthrax in an area of northern Italy.

Three cases of cutaneous anthrax are reported which occurred in a farming family in northern Italy. Epidemiological studies revealed contact with an infected cow (delivery of a stillborn fetus and slaughter). The cow was slaughtered soon after the delivery; cultures of carcass specimens yielded growth of Bacillus anthracis. The origin of the animal infection was not known. Serum samples were obtained from all 11 members of the family group and randomly from 10 of the 75 cows on the farm, which appeared to be in good health. Tests for antibodies against protective antigen and lethal factor using EIA and Western blot techniques were positive in three subjects (in paired sera) with cutaneous anthrax and in one subject who neither had had direct contact with the infected cow nor showed any sign of anthrax.


Erysipelas-like skin lesions associated with Campylobacter jejuni septicemia in patients with hypogammaglobulinemia.

Three cases are reported of hypogammaglobulinemic males with recurrent Campylobacter jejuni septicemia and erysipelas-like cellulitis without diarrhoea. In one patient Campylobacter jejuni grew from skin biopsy specimens. The findings in another patient were strongly suggestive of osteomyelitis caused by Campylobacter jejuni. Since the susceptibility of hypogammaglobulinemic patients to infection with Campylobacter jejuni is probably related to a lack of serum bactericidal activity against Campylobacter jejuni due to lack of IgM, two patients in whom previous antimicrobial treatment failed were treated with plasma infusions. This regimen supplemented with imipenem resulted in cure of these relapsing infections. Campylobacter jejuni septicemia must be considered in hypogammaglobulinemic patients who present with periodic fever and cellulitis.


Prosthetic joint infection due to Candida parapsilosis in the UK: case report and literature review.

Candida infection of joint replacements is a rare but increasingly reported phenomenon. A case of Candida parapsilosis prosthetic knee joint infection occurring in the UK is described. Cure followed removal of the prosthesis and treatment, first with a combination of amphotericin and 5-fluorocytosine, then ketoconazole.


Improved detection of HIV p24 antigen in serum after acid pretreatment.

HIV-1 p24 antigen was detected in 554 sera (509 from HIV-1 seropositive individuals and 45 sera from seronegative controls) using a conventional method with acid pretreatment of the sample in order to separate the p24 antigen/anti-p24 antibody immune complexes. In asymptomatic individuals there was a substantial increase in antigen detection (48.2% vs 8.4%). Similar results were also observed in ARC (59.1% vs 12.2%) and AIDS patients (85.7% vs 37.1%). It can be concluded that the acid treatment improves the sensitivity of conventional techniques to detect HIV-1 p24 antigen.


Pharmacokinetics of loracarbef and interaction with acetylcysteine.

A study was conducted to evaluate the pharmacokinetics of loracarbef, a new synthetic oral carbacephem antibiotic, following administration of 400 mg in normal male volunteers. The influence of food and possible interaction with acetylcysteine, a commonly used mucolytic agent, was also studied. Twelve healthy volunteers participated in the study and randomly received an oral dose of 400 mg loracarbef in the fasting state, 400 mg loracarbef following a standard breakfast or 400 mg loracarbef together with 200 mg acetylcysteine in granular form. Serum and urine concentrations were determined over 24 h by means of a bioassay. Loracarbef was well tolerated. Four volunteers complained of mild, transient headache. The substance was well absorbed with a mean peak level of 19.21 +/- 3.94 mg/l in the fasting state; it was primarily excreted in active form via the kidneys (urine recovery/24 h: 86-92%). The elimination half-life ranged from 70.3 to 102.0 min. Acetylcysteine had no effect on the absorption of loracarbef. The intake together with food delayed the absorption time, but had no influence on the bioavailability.


Comparative in vitro activity of clarithromycin. Spanish Collaborative Group.

The activity in vitro of clarithromycin, a new macrolide, was compared to that of various antibiotics in tests using 3,880 clinical isolates. Clarithromycin was two times more active than erythromycin against Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, streptococci of groups C, G and F, Brucella melitensis, Legionella pneumophila and Mycoplasma spp., 16 times more active against Ureaplasma urealyticum and 2 to 4 times less active against Campylobacter spp. In general, clarithromycin showed intrinsic activity 2 to 4 times higher than that of roxithromycin and 4 to 8 times higher than that of miocamycin. Cross-resistance was found between the macrolides. Clarithromycin was bactericidal against Streptococcus spp. and Haemophilus influenzae.


Lysosomal acid phosphatase is internalized via clathrin-coated pits.

The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed that at various stages of invaginating coated pits LAP colocalized with clathrin and plasma membrane adaptors (HA-2 adaptors). Quantitation of the immunogold label showed similar density of wild-type LAP in coated over non-coated areas of the plasma membrane, whereas an internalization-deficient, truncated mutant of LAP which lacks the cytoplasmic tail was less efficiently included into coated pits. Internalization of anti-LAP antibodies into endosomal vesicles was accompanied by rapid dissociation of the coat proteins as shown by an immunofluorescence assay. The role of clathrin-coated vesicles in internalization of LAP was further corroborated by microinjecting monoclonal antibodies against clathrin or HA-2 adaptors into BHK-LAP cells. Internalization of LAP as detected by an immunofluorescence assay was transiently blocked by microinjected antibodies against clathrin or HA-2 adaptors, whereas unrelated antibodies did not affect internalization. These data suggest that LAP is included into clathrin-coated pits of the plasma membrane for rapid internalization.


A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein.

A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells has been developed. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. NBD-cholesterol linoleate (NBD-CL) and octadecyl rhodamine B (R18) were incorporated simultaneously into LDL, as a RET donor and a RET acceptor, respectively. In this preparation of LDL (RET-LDL), efficient RET was observed, and after the disruption of the LDL particle by a Triton X-100 treatment, the relief of the RET was observed. RET-LDL was endocytosed by CHO cells via LDL receptors, and the RET-LDL particles were disintegrated after the uptake. The resultant relief of the RET upon the disintegration of the LDL was monitored by flow cytometry, and the amount of intact LDL in cells was estimated by calculation. The disintegration occurred with an about 25 min lag, and was inhibited by several lysosomal inhibitors. These results indicate that the disintegration was not a nonspecific event, but took place at the level of lysosomes. Since living cells can be analyzed by the present method, when coupled to flow sorting, it would permit the isolation of cells having different properties in the endocytic pathway of LDL.


Immunolocalization of the intermediate filament-associated protein plectin at focal contacts and actin stress fibers.

The distribution of plectin in the cytoplasm of Rat1 and glioma C6 cells was examined using a combination of double and triple immunofluorescence microscopy and interference reflection microscopy. In cells examined shortly after subcultivation (less than 48 h), filamentous networks of plectin structures, resembling and partially colocalizing with vimentin filaments, were observed as reported in previous studies. In cells kept attached to the substrate without growth for periods of 72 h to 8 days (stationary cultures), thick fibrillary plectin structures were observed. These structures were located at the end of actin filament bundles and showed co-distribution with adhesion plaques (focal contacts), vinculin, and vimentin. Only relatively large adhesion plaques (dash-like contacts) were decorated by antibodies to plectin, smaller dot-like contacts at the cell edges remained undecorated. Moreover, in stationary Rat1 cells plectin structures were found to be predominantly colocalized with actin stress fibers. However, after treatment of such cells with colcemid, plectin's distribution changed dramatically. The protein was no longer associated with actin structures, but was distributed diffusely throughout the cytoplasm. After a similar treatment with cytochalasin B, plectin's association with stress fibers again was completely abolished, although stress fibers were still present. The association of plectin with focal contact-associated intermediate filaments was demonstrated also by immunogold electron microscopy of quick-frozen, deep-etched replicas of rat embryo fibroblasts. These data confirm previous reports suggesting a relationship between intermediate filaments on the one hand, and actin stress fibers and their associated plasma membrane junctional complexes, on the other. Furthermore, the data establish plectin as a novel component of focal contact complexes and suggest that plectin plays a role as mediator between intermediate filaments and actin filaments.


Circadian rhythm of heat resistance in cotton seedlings: synthesis of heat-shock proteins.

Cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 12:12 h at 33 degrees C showed rhythmic changes in their resistance to heat shock of 53 degrees C for 40 min. The resistance was maximal at the middle of the light period and declined toward the end of the light period. One more peak of resistance developed in the middle of the dark period and declined toward the end of the dark period. Rhythmic changes in heat resistance persisted under continuous light for 3 cycles, indicating a circadian control. Under continuous light only one phase of resistance developed, lasting from the middle of the subjective night to the middle of the subjective day. The major heat shock proteins (HSPs) synthesized upon 30-min exposure to 40 degrees C, 49 degrees C or 53 degrees C were of 115, 89, 73, and 19 kDa. Their rate of synthesis depended on the inducing temperature, on previous exposure to high temperature and on the time in the light-dark cycle. The time dependency of the induction of certain HSPs persisted under continuous light, indicating a circadian control. No positive correlations was found between the rhythmic changes in heat resistance and the rhythmic changes in the synthesis of any HSP.


Low molecular mass heat-shock proteins of a light-resistant photoautotrophic cell culture.

Two low molecular mass heat-shock proteins (HSPs) of photoautotrophic cell culture (Chenopodium rubrum) have been cloned, sequenced and compared to published sequences. One of these HSPs (23.3 kDa) is posttranslationally transported into chloroplasts and shares homology with the other heat-shock proteins in the last third C-terminal region of the protein, but has a relatively unique sequence in the other two thirds. The correspondent small cytosolic protein of 18.3 kDa is related to all known small HSPs but has a unique DNA-binding domain that has not been described so far in the group of small cytosolic HSPs, it might represent a HSP which is translocated into the nucleus.


Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.


Epidermal growth factor receptor in chronic bile duct obstructed rats: implications for maintenance of hepatocellular mass.

Changes of the number and properties of the epidermal growth factor (EGF) receptor occur during liver regeneration and may be of importance in the maintenance of hepatocellular mass in liver cirrhosis. We therefore studied the changes in the number and distribution of EGF receptor in the development of liver cirrhosis induced by bile duct ligation. Receptor binding assays demonstrated a marked decrease in the binding capacity of crude plasma membrane fractions from 45 +/- SD 16 to 19 +/- 10 fmol/mg protein (p < 0.001) in control and bile duct ligated livers, respectively while the Kd increased after 3 days of bile duct ligation from 0.5 +/- 0.2 to 1.4 +/- 0.6 nmol/l. Total receptor concentration in the same membrane fractions, as assessed by Western blot analysis, was not changed. The expression of EGF receptor mRNA was reduced to about one third of control levels after 28 days of bile obstruction. Immunohistochemistry, performed using monoclonal antibodies against EGF receptor, showed a strong labeling of cytoplasm (87 +/- 3% positive) and plasma membranes (84 +/- 24%) but no labeling of nuclei in control livers. In bile duct ligated rats, in contrast, cytoplasmic staining was decreased (15 +/- 12%) already after 3 days of bile obstruction; labeling of canalicular membranes and nuclei appeared after 14 days. The shift of EGF receptor from plasma membranes to nuclei supports the notion that EGF receptor is involved in the maintenance of hepatocellular mass in this model of liver cirrhosis. This concept is supported by the finding of decreased mRNA for EGF receptor presumably representing down-regulation as seen in regenerating rat liver.


Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.


Effects on water diffusion of inhibitors affecting various transport processes in human red blood cells.

The water permeability of human red blood cells has been monitored by nuclear magnetic resonance (NMR) following exposure to inhibitors of various transport processes across their membranes. No significant inhibition of water diffusion could be detected after the treatment of red blood cells with the anion exchange transport inhibitor dihydro-4,4'-diisothiocyano-stilbene-2,2'-disulfonate (H2DIDS) or the glucose transport inhibitors diallyl-diethyl-stilbestrol (DADES), cytochalasin B, or 30 mM iodoacetamide. It is for the first time that the effects of glucose transport inhibitors has been studied in detail by the NMR approach. A special case proved to be phloretin, an inhibitor of anion, nonelectrolyte and glucose permeability. A small but statistically significant inhibition of water permeability (around 12% at 20 degrees C) was induced by exposure to 2 mM phloretin (for 60 min at 37 degrees C); after a pretreatment of cells with 12 mM N-ethylmaleimide (NEM), for 60 min at 37 degrees C, the degree of inhibition induced by phloretin increased (becoming 17% at 20 degrees C). None of the inhibitors prevented or potentiated the strong inhibitory effect on water diffusion of a mercurial, p-chloromercuribenzene sulfonate (PCMBS). No increase in the activation energy of water diffusion occurred by treatment with the reagents used (exception the effect of PCMBS). The present results clarify some conflicting reports concerning the effects on water permeability of inhibitors of various transport processes in red blood cells and indicate that in addition to the drastic inhibition induced by mercurials other reagents may also have inhibitory effects.


Distribution and metabolism of fluorescent sphingosines and corresponding ceramides bearing the diphenylhexatrienyl (DPH) fluorophore in cultured human fibroblasts.

Fluorescent D-erythro-sphingosines bearing the diphenyl-1,3,5-hexatrienyl group (DPH) as fluorophore were synthesized for the first time. Two isomers, the DPH-4(E)- and DPH-4(Z)-sphingosine [(2S,3R)-2-amino-6-(p-(18-phenyl)-13,15,17(E,E,E)-hexatrienyl)phenylh ex- 4(E/Z)-en-1,3-diol], and the N-hexanoyl derivative of DPH-4(E)-sphingosine (C6-DPH-ceramide) were studied for their distribution and metabolism in cultured human skin fibroblasts. Both DPH-sphingosines (4-trans and 4-cis) were not significantly acylated to ceramide in living cells, but converted to ceramide in vitro by microsomal protein from mouse brain, although slower than natural D-erythro-sphingosine. DPH-4(Z)-sphingosine showed the same Km like D-erythro-sphingosine (155 microM), but had a lower Vmax value, 0.85 instead of 1.9 nmol/mgh. An even poorer substrate was DPH-4(E)-sphingosine with a Km of 220 microM and a Vmax of 0.81 nmol/mgh. In cultured human fibroblasts, C6-DPH-ceramide was rapidly anabolized mainly to sphingomyelin. In addition, small quantities of glucosylceramide were also formed. DPH-sphingosines were easily incorporated into plasma membranes of cultured fibroblasts and are likely to undergo flip flop since intracellular membranes also became labeled, when endocytosis was blocked at low temperature (7 degrees C). The N-hexanoyl-DPH-trans-sphingosine, C6-DPH-ceramide, like NBD-C6-ceramide (Lipsky, N. G., R. E. Pagano: Science 228, 745-747 (1985)) labeled intracellular membranes at 7 degrees C and predominantly Golgi membranes at 37 degrees C. Like NBD-C6-ceramide (Pagano, R. E., M. A. Sepanski, O. C. Martin: J. Cell Biol. 109, 2067-2079 (1989)) the C6-DPH-ceramide also stained the Golgi complex in prefixed cells whereas DPH-trans- and DPH-cis-sphingosine did not, indicating that it is the ceramide structure rather than the fluorophore itself which is responsible for this staining. DPH-sphingosine opens a way for chemical synthesis of DPH-glycolipids and DPH-sphingomyelin which would well serve as donors in fluorescence energy transfer experiments to study possible sphingolipid clustering in biological membranes.


Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.


Interactions between thrombospondin and the small proteoglycan decorin: interference with cell attachment.

Decorin, a ubiquitous small interstitial dermatan sulfate proteoglycan, interacts with several extracellular matrix components, e.g., with type I collagen and fibronectin. Using a solid phase assay it is shown that the intact proteoglycan as well as its glycosaminoglycan-free core protein exhibits with KD values of about 5 nM and 2 nM, respectively, high affinity binding also to thrombospondin. However, the polysaccharide chain was required for an interaction with Sepharose-bound thrombospondin and served itself as ligand. In light of the results of binding studies with an N-terminal heparin-binding fragment of thrombospondin it is concluded that several structural features of thrombospondin and of decorin contribute to the mutual interaction of the two macromolecules. Thrombospondin substrata allowed attachment but prevented spreading of human skin fibroblasts. The addition of decorin or of its glycosaminoglycan-free core protein led to a considerable delay of cell attachment on a thrombospondin substrate. The strength of cell attachment appeared to be reduced. These data support the antiadhesive role of decorin regardless of whether subsequent cell spreading is supported or not.


Density-dependent accumulation of basic fibroblast growth factor in the subendothelial matrix.

Recent evidence indicates that basic fibroblast growth factor (bFGF), which lacks a conventional signal recognition sequence, is a component of the subendothelial matrix. However, the molecular mechanisms regulating its cellular release and subsequent matrix deposition remain equivocal. To examine the cellular and subcellular mechanisms regulating bFGF release and subendothelial sequestration, we generated polyclonal antibodies against a chemically cross-linked bFGF. We then used anti-bFGF IgG in conjunction with 3T3 cell [3H]thymidine incorporation assays, enzyme immunoassays and immunofluorescence to learn whether bFGF accumulation in the subendothelial matrix is dependent upon endothelial cell (EC)-cell contact, which coincides with growth arrest. In contrast to subconfluent cultures, which lacked any detectable extracellular matrix bFGF localization, bovine aortic and microvascular EC plated at confluent densities displayed a punctate extracellular staining pattern that was abolished when EC were pretreated with 10 micrograms/ml cycloheximide. Additionally, when EC were treated with either 1 mM beta-D xyloside, an inhibitor of proteoglycan assembly, or 100 micrograms/ml heparin, there was a 40% reduction in matrix-associated bFGF (quantified by image analysis of antibody stained cultures). 3T3 [3H]thymidine incorporation assays indicated that the beta-D xyloside-induced reduction of matrix-associated bFGF coincided with a significant increase in bFGF activity in the conditioned media. Neither sparsely-plated nor confluent EC cultures possessed specific bFGF localization of the nuclear compartment when cells were fixed using cold methanol; however, when EC were fixed in formaldehyde and lysed in isotonic buffers containing 0.1% Triton X-100 or absolute acetone, there was a marked decrease in anti-bFGF staining of the postconfluent extracellular matrix and a concomitant increase in nuclear fluorescence. Because bFGF-stimulated vascular cell growth has been implicated in controlling neointimal cell proliferation, we screened normal and atherosclerotic coronary blood vessels for bFGF, but we were unable to detect it either in lesioned or normal intima. In contrast, significant bFGF levels were observed in association with the EC and mesangial cells of the renal corpuscle, where heparan sulfate accumulates within the glomerular basement membrane. Our in vitro results suggest that bFGF accumulates within the proteoglycan-containing subendothelial matrix concomitant with the formation of cell-cell contacts. In situ, the composition of the microvascular matrix and the cellular phenotype may facilitate the selective accumulation of bFGF that we observed. This, in turn, may influence vascular morphogenesis and remodeling during angiogenesis.


The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A.

TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network, is utilized to study both the structure and function of the trans-Golgi network (TGN). The effects of brefeldin A (BFA) on the TGN were studied in comparison to its documented effects on the Golgi cisternae. During the first 30 min of BFA treatment, the TGN loses its cisternal structure and extends as tubules throughout the cytoplasm. By 60 min, it condenses into a stable structure surrounding the microtubule-organizing center. By electron microscopy, this structure appears as a population of large vesicles, and by immunolabeling, most of these vesicles contain TGN38. TGN38 cycles to the plasma membrane and back, which is shown by addition of TGN38 luminal domain antibodies directly to cell culture media. This results in rapid uptake of antibodies which label the TGN within 30 min, both in its native and BFA-induced conformation. A number of transmembrane proteins have been shown to take this cycling pathway, but TGN38 is unique in that it is the only one predominantly localized to the TGN. To investigate the cycling of TGN38, the endocytic pathway was labeled by internalization of Lucifer Yellow, and in the presence of BFA there was partial colocalization with TGN38. Further studies were carried out in which microtubules were depolymerized, resulting in dispersal of Golgi elements and inhibition of transport from endosomes to lysosomes. TGN38 cycling continues in the absence of microtubules. Taken together, these studies indicate that TGN38 returns from the plasma membrane via the endocytic pathway. We conclude that the TGN is structurally and functionally distinct from the Golgi cisternae, indicating that different molecules control membrane traffic from the Golgi cisternae and from the TGN.


The prevalence of Helicobacter pylori serum antibodies in children with recurrent abdominal pain.

As part of a large, prospective study we investigated the prevalence Helicobacter pylori serum antibodies in children with recurrent abdominal pain (RAP). All patients suffered from recurrent bouts of abdominal pain for at least 6 months and ranged in age from 6 to 12 years. H. pylori antibodies were detected using an enzyme-linked immunosorbent assay. The prevalence of H. pylori antibodies in the RAP group was compared to that of a control group which consisted predominantly of pre-operative children. None of the control group suffered or had suffered from RAP. Antibodies to H. pylori were found in 7 of 82 (8.5%) RAP patients and in 2 of 39 (5.1%) control children. The latter difference is not significant and suggests that RAP is only rarely caused in children by H. pylori infection.


Chlorinated hydrocarbons in the bone marrow of children: studies on their association with leukaemia.

Concentrations of chlorinated hydrocarbons and polychlorinated biphenyls (PCB) were determined by capillary column gas chromatography in samples of bone marrow from 38 children with leukaemia (16 samples/pools) and 15 control (5 pools). The highest mean and median concentrations were detected for total PCB (mean = 3.568 mg/kg fat basis/median 2.904 mg/kg) followed by the sum of the dichlorodiphenyltrichloroethane metabolites (1.775/1.059 mg/kg), hexachlorobenzene 0.354/0.260 mg/kg), the sum of the hexachlorocyclohexane isomers (0.133/0.093 mg/kg) and dieldrin (0.109/0.063 mg/kg). The CHC and PCB concentrations in bone marrow were two- to threefold higher than in fat tissue. Comparing children with and without leukaemia similar concentrations of CHC and PCB were found.


Chronic granulomatous disease 100% corrected by displacement bone marrow transplantation from a volunteer unrelated donor.

A boy whose chronic granulomatous disease (CGD) manifested in infancy, and whose elder brother had died at 7 years of age, had phagocytes with complete lack of functional cytochrome B-245 and which could not be induced by interferon gamma to achieve adequate staphylococcal killing. He underwent an elective displacement bone marrow transplant from a volunteer unrelated donor at the age of 8 months. This has achieved 100% replacement of the CGD granulocytes by those of the normal volunteer and the boy has since had a normal childhood for 3 years. Six previous transplants for CGD are briefly reviewed and illustrate that the host abnormal marrow must be completely displaced using an adequate dose of busulphan to ensure 100% stable engraftment of the donor's marrow and that this is best done under elective conditions before septic foci and irreversible organ damage have occurred. Criteria need to be developed to identify early those patients likely to have severe morbidity.


Insulin-dependent diabetes mellitus and severe atopic dermatitis in a child with adenosine deaminase deficiency.

We report a 2.3-year-old girl with complete lack of adenosine deaminase (ADA) activity who presented with severe atopic dermatitis and insulin-dependent diabetes mellitus but only mild recurrent infections. Abnormalities of immune function included profound depletion of CD8+ lymphocytes, hyperimmunoglobulinaemia E, and very low in vitro proliferative response to mitogens. Treatment with polyethylene glycol-conjugated ADA was followed by rapid amelioration of clinical and immunological conditions. The immunological and clinical features of this child suggest that the clinical spectrum of ADA deficiency may be broader than originally supposed.


Different syndromes associated with B19 parvovirus viraemia in paediatric patients: report of four cases.

The broad spectrum of clinical manifestations associated with B19 parvovirus often allows the infection to go unrecognized. We tested for the presence of B19 parvovirus and the specific serological response in serum from hospitalized patients submitted for viral investigations without any specific indications for B19 parvovirus. We diagnosed human parvovirus B19 infection in four paediatric patients showing different clinical manifestations. The patients, aged between 5 and 8 years, were admitted to hospital for: (1) petechial rash; (2) mononucleosis-like syndrome; (3) neurological syndrome; and (4) respiratory disease (in an immunodeficient patient).


Biochemical diagnosis and outcome of 2 years treatment in a patient with combined methylmalonic aciduria and homocystinuria.

We describe a patient with methylmalonic aciduria and homocystinuria due to a defect in cobalamin metabolism of the Cbl-C type mutant (McKusick 277400). Our case was diagnosed within the first 2 months of life by amino acid analysis (ion-exchange chromatography) and by biochemical studies in cultured fibroblasts ([14C]propionate incorporation, methionine and serine formation). We discuss the clinical course and the biochemical evolution after 2 years of hydroxycobalamin treatment that led to an improvement in general clinical condition and neurological performance.


Osteopetrosis: brain ultrasound and computed tomography findings.

A case of osteopetrosis presenting with rare features of dysmorphism with proptosis due to hypoplasia of the orbits and the temporal bone is described. The case also had calcifications in the periventricular regions, the falx cerebri and the corpora colliculi. These features and sonographic findings of osteopetrosis have not been reported previously. The sensitivity and specificity of imaging modalities in the diagnosis of intracranial calcifications is discussed.


Ultrastructure and immunocytochemistry of hepatic peroxisomes in rhizomelic chondrodysplasia punctata.

Peroxisomes were studied in the liver of two rhizomelic chondrodysplasia punctata patients using electron microscopy and catalase cytochemistry. Immunoelectron microscopy was carried out on the liver of one of these patients using antibodies to catalase, acyl-CoA oxidase, bifunctional protein, 3-ketoacyl-CoA thiolase and a 68 kDa peroxisomal membrane protein, in conjunction with protein-A colloidal gold. Moderately to markedly enlarged, flocculent peroxisomes were found in both patients. In one patient they were very heterogeneous with regard to the number per hepatocyte. The peroxisomes had very low levels of catalase as indicated by cytochemistry and immunocytochemistry. The three beta-oxidation enzymes were localised normally within the peroxisomes. The 68 kDa membrane protein was localised to the peroxisomal membranes. Some extra membrane loops were also identified using this antibody.


Craniofrontonasal dysplasia.

We report on nine patients with craniofrontonasal dysplasia (CFND). Seven classical cases had facial features suggestive of frontonasal dysplasia and coronal craniosynostosis. Extracranial abnormalities such as brittle nails with prominent longitudinal grooves or syndactyly of fingers and toes were observed in individual patients. In two families the father of classical cases showed a milder pattern of abnormalities, consistent with the diagnosis. We present a 2- to 13-year follow-up on our patients. Hypotonia and laxity of joints are common and may necessitate supportive measures. Mild developmental delay was noted in three out of six classical cases studied in detail. Unlike almost all other X-linked disorders, clinical expression in CFND is generally much more severe in females than in males. In contrast to previous reports of this condition, one of our severely affected cases is a male.


Evaluation of triggering systems for patient triggered ventilation for neonates ventilator-dependent beyond 10 days of age.

The performance of two triggering systems was compared during patient triggered ventilation (PTV) of infants ventilator-dependent beyond 10 days of age. Ten infants were studied who had a median gestational age of 26.5 weeks and a postnatal age of 15.5 days. PTV was administered via the SLE ventilator and the two triggering systems, an airway pressure monitor and the MR10 respiration monitor, were used in random order each for 30 min. The airway pressure trigger had a superior performance in that, although it did not differ significantly in delivered inflation volume or sensitivity to the MR10 respiration monitor, it had a shorter trigger delay (P < 0.01). Oxygenation improved in eight of the ten infants on the airway pressure trigger, but only in three on the MR10 respiration monitor. The reduction in PaCO2 was greater during PTV with the airway pressure trigger compared with the MR10 respiration monitor (P < 0.01). We conclude that the airway pressure trigger has a superior performance compared to the MR10 respiration monitor trigger in infants who are ventilator-dependent beyond 10 days of age.


Dynamic lung inflation during high frequency oscillation in neonates.

The effects of high frequency oscillation (HFO) on dynamic lung inflation were examined in 22 neonates ventilated for respiratory disease. HFO was combined with conventional ventilation and a series of frequencies from 2-25 Hz was tested. Dynamic lung inflation was measured using a jacket plethysmograph which was converted to a measure of alveolar pressure using the compliance of the respiratory system obtained during conventional ventilation. The results showed an increase in dynamic lung inflation with frequency such that volume increased by 0.4 ml for each increase of 10 Hz. Alveolar pressure increased by 1.2 cm H2O for each increase of 10 Hz. Dynamic lung inflation also increased with increased volumes of oscillation.


Exchange transfusion: evaluating the use of a mixture of citrated red cells and heparinized plasma.

In 1987 a mixture for exchange transfusion was introduced in the Netherlands. It was composed of citrated red cells, heparinized plasma and third-party platelets if necessary. Selected biochemical and haematological properties of this mixture were compared to fresh heparinized whole blood, which was at that time the blood product of choice for exchange transfusion. The parameters of the mixture were less physiological than fresh heparinized whole blood. In addition, retrospective analysis of the same parameters was performed upon 149 blood samples from newborn infants who had undergone exchange transfusion. The bilirubin decreasing capacity of the mixture was adequate. Most other parameters did not change considerably and remained within the physiological range. This mixture may be an adequate product for exchange transfusion. However, to be certain of its safety and suitability, several other biochemical and haematological aspects must be studied in addition to the immunological and infectious risks.


Catecholamine levels and associated cardiovascular responses in infants with meconium-stained amniotic fluid.

Catecholamine levels and associated cardiovascular responses were determined in 21 control and 30 term infants with meconium-stained amniotic fluid (MSAF). Cord arterial blood was obtained for determination for norepinephrine (NE) and epinephrine (EPI) levels; cardiovascular measurements included heart rate and systolic blood pressure at 30 min. Pregnancy was uncomplicated in all cases. The total length of labor 964 +/- 402 versus 555 +/- 312 min (P < 0.001) and active phase of labor 300 +/- 261 versus 135 +/- 104 min (P < 0.001) were significantly longer in MSAF infants when compared to controls. The 1 min Apgar score was < 7 (range 1-6) in 11 of 30 MSAF infants versus 0 of 21 control infants; these 11 infants required positive pressure ventilation for approximately 60 s secondary to transient respiratory depression (RD). The 5 min Apgar score was > 7 in 49 of the 51 infants. Infants with MSAF exhibited a higher PaCO2 (6.89 +/- 1.17 vs 6.24 +/- 1.17 mmHg; P < 0.02) and lower pH (7.25 +/- 0.06 vs 7.29 +/- 0.05; P < 0.02) compared to controls. Infants with transient RD demonstrated the most marked arterial blood gas differences, i.e., PCO2 7.41 +/- 1.30 mmHg (P < 0.001), pH 7.21 +/- 0.07 (P < 0.001) and base deficit -6 +/- 4 versus -3 +/- 2.8 (P < 0.001) when compared to controls. NE and EPI levels were significantly higher in MSAF versus control infants, i.e., 12600 +/- 3040 versus 3740 +/- 1000 pg/ml (SEM) (P < 0.02) and 1550 +/- 250 versus 620 +/- 130 pg/ml (P < 0.001) respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Familial cavernous angiomas of the brain: observations in a four generation family.

A family with a history of cavernous angiomas of the brain was investigated by MRI. The disease was present in four generations of the family and is consistent with autosomal dominant inheritance. Among affected individuals, there was considerable variability in the extent of intraparenchymal cavernomas and neurological symptoms as a result of bleeding events. Three siblings manifested with seizures, two affected persons were symptom free at the time of investigation, and one sibling had neurological symptoms without certain correlation with cavernomas. The disease appeared to have an earlier onset in younger generations.


Pediatric urinary diversion: review and own experience.

New insights into the diseases of childhood, profound improvements and new developments in surgical techniques as well as the knowledge gained from long-term follow-up have altered the strategies and indications for urinary diversion in childhood. Continent urinary diversion is generally the method of choice. We are able to construct high capacity, low pressure reservoirs to protect the upper urinary tract and achieve continence. Nowadays, there is hardly any indication for permanent cutaneous urinary diversion. Temporary and intermediate cutaneous diversion are used only when serious conditions such as renal function deterioration occur. The possibility to convert any incontinent form of urinary diversion into a continent form changed the status of intermediate diversion. The indications for urinary diversion, the procedures available, the operative technique, a literature review, our own experience, contemporary strategies and controversies are described and discussed.


Application of flexible renoureteroscope for antegrade urethroscopy in the treatment of congenital anterior urethral valve.

Antegrade urethroscopy was performed through a cystostomy using a 10.8-fr caliber flexible renoureteroscope in 4 children (aged 2 years and 8 months, 2 months, 14 years and 1 month) with anterior urethral valve. The anatomy of the valves as well as their destruction after transurethral resection (TUR) were well visualized by antegrade urethroscopy. This technique was helpful in performing TUR-valve, after which the findings of voiding cystourethrography in every case and the findings of uroflowmetry in the elder 2 cases showed remarkable improvement. This technique is relatively simple to perform and is thought to be one of the best methods to treat anterior urethral valve. This technique is also applicable to other lower urinary tract lesions in children.


Quantitation of urinary Tamm-Horsfall protein in children with urinary tract infection.

It has been suggested that urinary Tamm-Horsfall protein (THP) prevents colonization of the urinary tract by binding uropathogens. We tested the hypothesis that low urinary THP levels may predispose to urinary tract infection (UTI) by measuring THP levels in children. We studied a cohort of 35 girls with uncomplicated recurrent UTI (group 1) that was compared with 27 patients with myelomeningoceles undergoing clean intermittent catheterization (group 2) and 16 female controls (group 3). We measured urinary THP in both aggregated (aTHP) and disaggregated form (dTHP), leukocyte esterase activity, urine chemistries and culture. No significant differences in dTHP or aTHP levels were seen between groups 1 and 3, but group-1 patients had higher dTHP levels than group-2 patients (p < 0.008). History of reflux or the presence of bacteriuria or pyuria at the time of urine collection did not affect dTHP levels; in contrast, pyuria or bacteriuria at the time of sampling was associated with markedly lower aTHP levels when compared with sterile samples (p < 0.0001). For all groups, measured quantities of dTHP did not correlate with aTHP levels. We conclude that excretion of dTHP in children with history of recurrent UTI is not reduced. In contrast, concentrations of aTHP are profoundly depressed in children during times of UTI, suggesting a role for THP in the pathogenesis of UTI. Assaying THP in its aggregated form may prove valuable when studying its physiologic function and merits further investigation.


Renal cell carcinoma with extension into the inferior vena cava: problems in diagnosis, staging and treatment.

Management of renal cell carcinoma with extension into the vena cava is one of the highest challenges in urology. The delineation of the upper level of the endocaval thrombus, its possible extension into the hepatic veins and possible involvement of the caval wall are crucial points and dictate the operative strategy. From 1978 to 1989, at the Department of Urology of the University of Padua, 29 patients with renal cell carcinoma and caval extension were submitted to surgery. Seventeen patients were staged V2 according to the UICC TNM Staging System 1983, 2 patients V3 and 10 V4. The median patient age was 57 years and the male:female ratio was 1.9:1. A complete thrombus removal was accomplished in all patients. In 2 patients, a caval resection was necessary. Five patients died during the perioperative period. The patient survival rate was comparable to that of T3N0M0-staged cases. Survival rates at 1 and 4 years were, respectively, 96.8 and 40% for V2 cases, 100 and 100% for V3, 85.7 and 40% for V4. It seems, therefore, that in patients with locoregional disease and satisfactory conditions, the presence of vascular invasion did not lead to a worst prognosis. By itself, the extent of the caval thrombus seems not to influence the prognosis.


Prognostic significance of granular cell content in renal cell carcinoma.

The relation between survival and tumor cell type in renal cell carcinomas was reviewed in 79 cases. The mean follow-up period was 10 years. Tumors were classified into 5 groups according to the percentage of granular cells: group 1, 0-5%; group 2, 6-20%; group 3, 21-40%; group 4, 41-80%, and group 5, over 80%. Half of the patients (50.6%) revealed less than 5% of granular cells, and almost two thirds of the patients (64.64%) had less than 20% of granular cells. Our results showed a lack of correlation between the percentage of granular cell in the tumor and long-term survival (chi 2 p > 0.9 after 10 years). When the data were analyzed in groups of patients with the same clinical stage or nuclear grading, no significant correlation between the percentage of granular cells and long-term survival was found. However, the short-term survival, up to 3 years after diagnosis, was significantly higher for patients with tumors containing less than 20% of granular cells (64.6% in comparison to 46.4% survival for patients with over 20% granular cells; chi 2 p = 0.1). In addition, our data showed a significant correlation between the survival of patients and nuclear grading (chi 2 p < 0.003) as well as the surgical staging of the tumor (chi 2 p < 0.001).


Bilateral asynchronous renal cell carcinoma. Computed tomography of the contralateral kidney 10-43 years after nephrectomy.

Computed tomography (CT) was carried out in 31 patients 10-43 years after surgery for renal cell carcinoma, 10 belonging to a consecutive series of patients operated upon at one urological department 10 years previously. Twenty-eight patients were symptomless, and 3 had flank pain, severe fatigue and hematuria, respectively. Cancers in the remaining kidney were found 13-21 years after nephrectomy in 4 of 31 patients (12.9%). The 3 patients with symptoms were among these 4. An adenoma was found in 1 patient 10 years after nephrectomy. The cancers were treated by renal resection in 2 patients, multiple tumors made nephrectomy necessary in 1 patient and 1 patient was not operated upon because of disseminated disease. The adenoma indicated future checkup by CT. Three of the 4 new cancers had a dismal outcome. The renal parenchyma was found to be essentially normal in all the other 26 patients, irrespective of the widely varying time interval between nephrectomy and CT. Asynchronous bilateral renal cell carcinoma has a poor outcome which presumably can be improved by early diagnosis and aggressive treatment. CT is the method of choice for early detection and follow-up of renal tumors. It should be carried out every other year after nephrectomy for renal cell carcinoma.


Colorectal cancer involving the urinary tract.

The survival rate among patients with colorectal cancer classified as Dukes' B or C who underwent extended resection due to infiltration of neighboring structures is significantly lower than among patients in the same Dukes' stage without infiltration of adjacent structures. Seventy-one patients with rectosigmoid cancer (37 primary and 34 recurrent tumors) involving the urogenital tract were retrospectively analyzed. Patients with primary tumors had a markedly higher mean survival rate after radical surgery (60.7 months) than after palliative procedures (28.2 months), while no such difference was seen among patients with recurrent tumors (17.6 or 14.1 months, respectively). From the urologic point of view, radical intervention is mandatory in cases of primary rectosigmoid carcinoma involving the urogenital tract. Aggressive surgery for recurrent tumors appears questionable, however; in these cases, priority should be given to improving the quality of life, recommending palliative surgery as the preferable approach.


Surgical treatment of acute epididymitis and its underlying diseases.

Two hundred and seventy of 1,031 patients (26%) treated for acute epididymitis between 1979 and 1989 underwent surgery for their disease. Eighty percent of these interventions were therapeutic, 20% diagnostic to exclude testicular torsion or tumor. Diagnostic procedures detected underlying diseases that required surgery in 278/1,031 patients (27%). Two hundred and sixteen of these diseases caused lower urinary tract obstruction and were potentially responsible for the acute epididymitis. There were striking differences in type and frequency of the surgery performed depending on the patients' age. Surgery of epididymitis was almost exclusively diagnostic in patients of a young age (exclusion of testicular torsion) and almost always therapeutic in patients of an old age (abscess, prolonged disease). The rate of surgery for underlying diseases was the same for the young (55%) and old (62%) age groups. Obstruction of the lower urinary tract was the predominant cause for surgical treatment of underlying diseases, being mainly due to malformation in the young age group and due to acquired obstructive diseases in the old.


Image analysis in the determination of ploidy and prognosis in renal cell carcinoma.

The usefulness of ploidy determination by flow cytometry in renal cell carcinoma is disputed. An alternative technique for DNA quantitation, computerised static image analysis, has several advantages over flow methods. Tissue from 90 cases of primary renal cell carcinoma was analysed by image analysis. Cases were assigned a histological grade and staged and stratified by the TNM system. Fifty-two cases were diploid, 28 were aneuploid and 9 were tetraploid (1 was unanalyzable). Heterogeneity of ploidy was noted in 4 cases. The degree to which nuclear DNA content exceeded that of normal nuclei from the same block was also calculated (5C exceeding rate). Stage and grade provided independent prognostic information. Aneuploidy was associated with higher mortality but neither ploidy nor 5C exceeding rate were prognostic indicators. Image analysis is a useful and accurate method for ploidy determination and provides additional data on DNA quantitation but this information is not predictive of the clinical outcome.


Muscle regeneration after endoureteropyelotomy?

Three specimens of ureteropelvic junction, obtained at dismembered pyeloplasty after successful endoureteropyelotomy, were studied. In the regenerative tissue, numerous cells were found with morphological, immunohistochemical and ultrastructural characteristics of mature smooth muscle cells; no regeneration of bundle arrangement was observed.


Human penile hemodynamics by Xenon-133 clearance method in physiological erection.

We measured penile blood flow during physiological erection using the Xenon-133 clearance method in order to diagnose the exact cause of impotence. In 16 patients, in whom complete erection was obtained after visual sexual stimulation, the mean penile blood flow during erection was apparently higher than that in the flaccid state. On the contrary, in 6 patients whose reaction to visual stimulation was poor, penile blood flow did not change before and after stimulation. We considered that increased arterial flow was indispensable, but complete venous outlet obstruction was not necessary to produce a rigid erection. In addition, in the flaccid state the mean penile blood flow in the poor response group was apparently higher than that in the response group. This means that some patients who fail to obtain sufficient erection after visual sexual stimulation have increased venous outflow from the corpus cavernosum in the flaccid state. Based on these findings, the main cause of erectile dysfunction in such cases is probably attributed to disorders of the venous outlet system in the cavernous body.


Laparoscopic nephrectomy in a porcine model.

Laparoscopic nephrectomy was performed in 15 male pigs, the procedure was successful in 14. Extraction of the intact kidney through a 5-cm lower abdomen incision was done in 7 animals; complete destruction and evacuation of the kidney was accomplished by a round-knife suction device through a 1-cm port in another 7 pigs. Grossly, the specimen consisted of sausage-like tubular renal tissue and a small amount of tissue debris. Pathology revealed that the glomerular and tubular structures were well preserved, no interstitial hematoma could be found. Four ports were usually used, one 1-cm umbilical camera port, one 0.5-cm port for ureter traction, and two 1-cm working ports along the midclavicular line. All the pigs recovered uneventfully. The average operation time was 3 h 20 min. The application of endo-GIA (United States Surgical Corporation) for renal hilum reduced the operative time to 2 h 20 min. Complications included renal vein tear during endoclip application and cutting in the first case, mild subcutaneous emphysema in 2 cases. This first pig received exploratory laparotomy for the repair and ligation of the renal vein. No more major complications occurred with increasing experience. From this porcine experiment, we conclude that the combination of laparoscopy, a tissue destroyer and an endobag for the entrapment of kidney seem to be a promising technique for clinical nephrectomy.


The appendix as a continence mechanism.

Although we have progressed very well in creating large capacity, low pressure reservoirs, the construction of a simple and reliable continent outlet still remains a problem. The appendix vermiformis serves well as a continence mechanism for either the bladder or intestinal reservoirs for urine. The different surgical techniques described in the literature are reviewed and discussed in this context. Moreover, we report on our clinical and experimental results of using the appendix during the Mainz pouch procedure for continent urinary diversion.


Hyperbaric oxygen therapy for control of intractable cyclophosphamide-induced hemorrhagic cystitis.

We report a case of intractable hemorrhagic cystitis due to cyclophosphamide therapy for Wegener's granulomatosis. Conservative treatment, including bladder irrigation with physiological saline and instillation of prostaglandin F2 alpha, failed to totally control hemorrhage. We then used hyperbaric oxygen at an absolute pressure of 2 atm, 5 days a week for 8 consecutive weeks. The bleeding ceased completely by the end of treatment and the patient remained free of hematuria thereafter. No side effect was noted during the course of therapy. In future, this form of therapy can offer a safe alternative in the treatment of cyclophosphamide-induced hemorrhagic cystitis.