Tobramycin once- vs thrice-daily for elective intravenous antipseudomonal therapy in pediatric cystic fibrosis patients.

PURPOSE: We hypothesized that a single intravenous (iv) tobramycine infusion (treatment B) would have equivalent anti-infectious efficacy in chronic Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) as the commonly performed treatment of three doses (treatment A) . Toxicity and practicability may even be improved in the single-dose regimen. METHODS: This was a randomized crossover study comparing outcome after 14 and 35 days. The primary end-point was a decrease in the leukocyte count, and the secondary end-points were clinical and lung function parameters, Pseudomonas quantification in sputum, and inflammation markers (immunoglobulin G, C-reactive protein) in serum. 30 patients (20 female, mean age 11.2 years, mean age range 1.7-18.1 years) received elective 14-day courses of treatments A or B, followed by the alternative treatment after a mean interval of 37 (+/- 21) weeks. RESULTS: With the exception of PA density, there were no significant differences between both treatment strategies after 14 days of treatment. After 35 days of treatment, there were no significant changes in the leukocyte count and inflammation markers. Both treatment strategies reduced the bacterial load in the airways, as reflected by a decreased PA density in sputum. Nephrotoxicity was equal in both groups, with a transient slight elevation of urinary N-acetyl-beta-glucosaminidase concentrations. Standard audiometry tests revealed no evidence of a hearing impairment in any patient following therapy. Mean body weight increased during the study period by 0.5 kg. Forced expiratory volume increased by approximately 5% of the predicted volume, forced vital capacity increased by 2% of predicted capacity, and forced mid expiratory flow rate increased by 7% (A) or 4% (B) of the predicted normal value, although these changes were not statistically significant. CONCLUSION: We conclude that tobramycin given in a daily single dose (with the advantage of being more practical in a home environment) has an efficacy equal to that of three daily doses in terms of elective antipseudomonal therapy in clinically stable patients with CF.

Continuous vs thrice-daily ceftazidime for elective intravenous antipseudomonal therapy in cystic fibrosis.

BACKGROUND/AIM: We hypothesized that a continuous 24-h infusion of 100 mg/kg per day ceftazidime (treatment C) would result in equivalent or even superior anti-infectious efficacy in chronic Pseudomonus aeruginosa (PA) infection in patients with cystic fibrosis (CF) in comparison to the usual application of 200 mg/kg per day ceftazidime in three doses (treatment T). METHODS: This was a randomized crossover study comparing outcome after 14 days and 35 days. Tobramycin administered once daily (10 mg/kg per day) was administered concomitantly in both groups. The primary end-point was a decrease in the leukocyte count, and the secondary endpoints were clinical and lung function parameters, Pseudomonas quantification in sputum, and inflammation markers (immunogloblulin [Ig] G, C-reactive protein [CRP]) in serum. All patients received antibiotics electively as 14-day courses on a regular basis, not for acute exacerbations. RESULTS: Fifty-six patients (29 females, mean patient age 14.4 years, age range 5-37) initially received treatments C or T, followed by the alternative treatment after a mean interval of 37 (+/- 21) weeks. After 2 weeks of antibiotic treatment, the overall study group showed significant improvements compared to baseline for body weight, leukocyte counts, CRP, forced expiratory volume in 1 s (FEV(1)), FVC (forced vital capacity), and bacterial load in the airways, with no significant differences between treatment groups. Both regimens were well tolerated. Three weeks after cessation of antimicrobial therapy, leukocytes and PA density had returned to pre-treatment values. CONCLUSION: We conclude that continuous or thrice-daily dosing of intravenous ceftazidime, both combined with once-daily tobramycin, are equally effective application regimens for elective antipseudomonal therapy in clinically stable patients with CF.

Applicability of different antibodies for immunohistochemical localization of CFTR in sweat glands from healthy controls and from patients with cystic fibrosis.

The hereditary disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Understanding of the consequences of CFTR gene mutations is derived chiefly from in vitro studies on heterologous cell cultures and on cells hyperexpressing CFTR. Data from ex vivo studies on human tissue are scarce and contradictory, a fact which is in part explained by secondary tissue destruction in most affected organs. The purpose of this study was to establish conditions under which wild-type and mutated CFTR can be studied in affected human tissue. Sweat glands carry the basic defect underlying CF and are not affected by tissue destruction and inflammation. Therefore, we used this tissue to test a panel of eight different CFTR antibodies under various fixation techniques. The antibodies were tested on skin biopsy sections from healthy controls, from CF patients homozygous for the most common mutation, DeltaF508, and from patients carrying two nonsense mutations. Of the eight CFTR antibodies, only three-M3A7, MATG 1104, and cc24-met the criteria necessary for immunolocalization of CFTR in sweat glands. The labeling pattern in the CF sweat glands was consistent with the postulated processing defect of DeltaF508 CFTR. The antibodies exhibited different sensitivities for detecting DeltaF508 CFTR.

Pitfalls in the immunohistochemical localization of the cystic fibrosis transmembrane conductance regulator in paraffin embedded sweat glands.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the hereditary disease cystic fibrosis. The most frequent mutant deltaF508 has been shown in vitro to be retained in the endoplasmic reticulum. Ex vivo studies using immunohistochemical labelling in cryofixed skin biopsies have confirmed the mislocalization of deltaF508 CFTR in sweat glands. The purpose of this study was to test CFTR antibodies in paraffin-embedded skin biopsies to take advantage of the superior tissue preservation as compared to cryofixation. A panel of 7 CFTR antibodies was applied to skin sections of healthy controls and of cystic fibrosis patients homozygous for the deltaF508 mutation. Sweat gland labelling consistent with CFTR localization and different between control and cystic fibrosis tissue was obtained with 2 antibodies. Conventional staining controls confirmed the labelling specificity. The antibodies were subsequently tested in a series of 237 sections of 16 biopsy specimens. However, the sweat gland labelling pattern proved not to be dependent on CFTR genotype. This finding was the sole indicator of non-specificity of the staining which was revealed only by the size of our random sample. Our results emphasize that CFTR immunolabelling following formalin fixation has to be interpreted with the utmost caution.

DeltaF508 CFTR protein expression in tissues from patients with cystic fibrosis.

Heterologous expression of the cystic fibrosis transmembrane conductance regulator (CFTR) provided evidence that the major cystic fibrosis (CF) mutation DeltaF508 leads to defective protein folding in the endoplasmic reticulum, which prevents its processing and targeting to the cell surface. In this study, we investigated endogenous CFTR expression in skin biopsies and respiratory and intestinal tissue specimens from DeltaF508 homozygous and non-CF patients, using immunohistochemical and immunoblot analyses with a panel of CFTR antibodies. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells. In DeltaF508 homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas DeltaF508 CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wild-type by signal intensity or localization. The tissue-specific variation of DeltaF508 CFTR expression from null to apparently normal amounts indicates that DeltaF508 CFTR maturation can be modulated and suggests that determinants other than CFTR mislocalization should play a role in DeltaF508 CF respiratory and intestinal disease.

Geographic distribution and origin of CFTR mutations in Germany.

The geographic distribution and origin of CFTR mutations in Germany was evaluated in 658 three-generation families with cystic fibrosis (CF). Fifty different mutations were detected on 1305 parental CF chromosomes from 22 European countries and overseas. The major mutation. delta F508 was identified on 71.5% of all CF chromosomes, followed by R553X (1.8%), N1303K (1.3%), G542X (1.1%), G551D (0.8%) and R347P (0.8%). According to the grandparents' birthplace, 74% of CF chromosomes had their origin in Germany; the delta F508 percentage was 77%, 75%, 70% and 62% in northern, southern, western and eastern Germany, respectively. Ten or more mutant alleles in the investigated CF gene pool originated from Austria, the Czech Republic, Poland, Russia, Turkey and the Ukraine. This widespread geographic origin of CFTR mutations in today's Germany reflects the many demographic changes and migrations in Central Europe during the 20th century.

First case of disseminated Mycobacterium avium infection following chemotherapy for childhood acute myeloid leukemia.

A 14-year-old girl of Indian origin with acute myeloid leukemia (AML) is presented, who was diagnosed at the age of twelve. Antileukemic chemotherapy had to be discontinued after 6 weeks because of persistent high fever and the emergence of liver and spleen abscesses. Serologic and biopsy findings were consistent with disseminated candidiasis; however, a liver biopsy also revealed granulomatous lesions with caseous degeneration. No acid-fast bacilli could be detected. Upon antifungal treatment the patient's condition improved, but fever spells and high inflammatory blood parameters persisted. One year after the diagnosis of AML was established, Mycobacterium avium was cultured from bone marrow aspirates. The patient's cellular immunity was severely compromised at that time as reflected by the marked depression of T-lymphocyte counts, in particular of CD4-positive cells. HIV and other lymphotropic virus infections were subsequently excluded. After 5 months of specific treatment the patient recovered from mycobacterial infection and remains in first remission of AML. Opportunistic infections have rarely been diagnosed in oncologic patients to date, while data on T-cell function in AML is sparse. Fever of unknown origin should prompt the search for infectious agents unusual to date in this patient group.

[Paracetamol poisoning in infancy]. / Paracetamolvergiftung im Säuglingsalter.

An eight-week old infant with alcohol embryopathy, weighing 3,700 g, was found to have abnormal liver functions (GPT 312 U/l, Quick value 25%) after surgical repair of a stenosis of the left ureter at its origin. The hospital notes indicated that the infant had been given a total of 1.6 g paracetamol over 60 hours for postoperative restlessness and pain. The serum paracetamol level was 60 mg/l 8 hours after the last dose of the drug. Blood exchange transfusion lowered the paracetamol level to 11 mg/l within 14 hours. After the exchange transfusion further signs of poisoning, namely renal impairment and a severe encephalopathy were noted, and Candida was demonstrated in urine, tracheal secretion and ascites. The renal and hepatic damage proved reversible under symptomatic treatment. But the child, now 1 year old, is severely retarded mentally and in its motor functions. These sequelae may be a residue of the paracetamol poisoning, complications of the clinical course or a combination of the two.

Long term culture of nasal epithelial cells.

Cystic fibrosis (CF) basic research has been hampered by the lack of a suitable animal model and is therefore dependent on the availability of human tissue. To date, the basic defect remains unknown, however, over the last decade convincing data have been produced that link the defect to altered epithelial electrolyte transport. Moreover, recent results of DNA analysis could confirm that the putative CF gene product is indeed expressed in epithelial tissues. Unfortunately, epithelial cells are difficult to maintain in culture, especially transport-active epithelia tend to be short-lived. We have devised a simple method for the subculturing of nasal epithelium which has been shown to exhibit altered electrolyte transport in CF patients. Nasal epithelial cells were isolated from nasal polyps obtained at surgery by protease digestion. Subsequently, cells were plated in culture medium and irradiated mouse fibroblasts were added as a feeder layer after 48 h. After 5-15 d, epithelial cells could be subcultured without loosing their growth potential, and were continuously maintained in culture for up to 4 months. The cells were frozen in liquid nitrogen following standard methods. Growth could be reinstituted after thawing the epithelium. Subcultured and thawed cells were clearly characterized as of epithelial origin and distinguished from mesodermal cells by their reaction with antibodies against keratin, vimentin, and desmin.

The nasal polyps as a tool for basic research in cystic fibrosis.

Total RNA and mRNA were prepared from cystic fibrosis (CF) and control nasal polyps and nasal epithelial cells. Genomic clones from the chromosomal region of the CF locus were screened by northern blots. A representative cDNA library from nasal polyps was cloned in the vector lambda gt10. For the construction of a physical genomic map around the CF locus single gene markers were isolated from metaphase 1:7q2qter chromosomes by laser micro-dissection and subsequent microcloning. A linkage study with the polymorphic markers met-H, met-D, and pJ3.11 was performed in 53 German CF families with at least 2 children. No significant correlation of any haplotype on the CF chromosomes with the clinical severity of the course of the disease could be observed, which provides evidence that cystic fibrosis is genetically homogeneous.

Prenatal detection of cystic fibrosis; comparative study of maltase and alkaline phosphatase activities in amniotic fluid.

The potential value of microvillar enzymes in the prenatal diagnosis of cystic fibrosis (CF) has previously been demonstrated and is corroborated in the present comparative study. Maltase and alkaline phosphatase (ALP) activities were studied in the amniotic fluids of 57 pregnancies with a 1 in 4 risk for CF or with a known CF outcome and in 489 controls. A simple assay for maltase activity (MU-maltase) with the fluorogenic substate 4-methylumbelliferyl alpha-glucoside, offers great technical advantages and an at least equal detection rate of CF, when compared to the previously used test with maltose as substrate. Intestinal ALP was estimated either as phenylalanine inhibitable activity (PI-ALP) or as the proportions of residual activity in the presence of the inhibitors phenylalanine or homoarginine. MU-maltase and PI-ALP appeared the most successful methods: both tests were able to detect 14 of the 16 (88 per cent) pregnancies with fetal CF. Each of the two tests alone also allowed a correct prediction in 24 of the 25 pregnancies at risk but with normal outcome; however all 25 cases could be correctly predicted by a combined evaluation. It is suggested that more than one intestinal enzyme activity should be evaluated to allow optimal results in the prenatal monitoring of pregnancies at high risk for CF.

Characteristics of maltase activity in amniotic fluid.

The nature and origin of maltase activity present in amniotic fluid, and used as a marker enzyme in the prenatal monitoring of cystic fibrosis, has been studied. Using monoclonal antibodies against human intestinal disaccharidases and via heat inactivation experiments it is shown that the maltase activity found in amniotic fluids from pregnancies of 16-24 wk of gestational age originates completely from sucrase-isomaltase; no maltase-glucoamylase could be detected. With various monospecific antibodies the possible contribution of non-intestinal brush border enzymes to the total maltase pool could be excluded: neither renal nor lysosomal maltase appeared to be present.

Sucrase-isomaltase and cystic fibrosis.

The intestinal microvillar enzyme complex sucrase-isomaltase has been studied in cystic fibrosis and control ileum. A number of biochemical parameters of the enzyme in ileum homogenates have been determined. Both solubilized as well as membrane-bound sucrase-isomaltase were analyzed with respect to their reaction with monoclonal antibodies against human sucrase-isomaltase. Finally the subcellular localization of sucrase-isomaltase was verified by immunoelectronmicroscopy or via the analysis of purified brush-border membrane preparations. At all levels no significant differences could be detected between sucrase-isomaltase of cystic fibrosis and control ileum. It is concluded that an abnormal subcellular localization and/or abnormal enzymatic activity of sucrase-isomaltase in cystic fibrosis intestine cannot explain the markedly decreased disaccharidase activities in amniotic fluids from pregnancies resulting in a child affected with cystic fibrosis.