Prospective evaluation of emerging bacteria in cystic fibrosis.

BACKGROUND: Bacteria contribute considerably to the progression of lung disease in cystic fibrosis. In this prospective, multi-centre study, we aimed to evaluate the occurrence of emerging bacteria and the physicians' assessments of the clinical importance of these findings. METHODS: Twelve CF centres (total number of patients: 1419) reported the detection of any Stenotrophomonas maltophilia, Burkholderia cepacia complex, MRSA, Alcaligenes xylosoxidans, Klebsiella species and Mycobacteria during an observation period of 6 months. RESULTS: 213 specimens with emerging bacteria were reported from 145 different patients. The proportion of patients with emerging bacteria differed between centres (3-38%, mean: 12.6%) and increased with age. The predominant bacterium was S. maltophilia (n=106 positive specimens), followed by Klebsiellae (n=36), B. cepacia complex (n=31), A. xylosoxidans (n=16), Mycobacteria (n=11), MRSA (n=11), and others (n=2). In many instances the same microorganisms had already been reported earlier, indicating intermittent or chronic colonisation. The clinical status was reported to be stable in 70% of patients, and antibiotic treatment was anticipated for 46% of positive specimens. Comparison of clinical data to age matched controls did not reveal any significant differences with regard to pulmonary and nutritional status prior to detection of emerging bacteria. CONCLUSION: These data suggest a high variability between centres regarding the prevalence of emerging bacteria. Most patients maintained a stable clinical condition during the 6-month study period despite being colonised with emerging bacteria.

Diabetes mellitus and cystic fibrosis: comparison of clinical parameters in patients treated with insulin versus oral glucose-lowering agents.

The prevalence of cystic fibrosis-related diabetes melltitus (CFRD) is increasing as patients with cystic fibrosis (CF) live longer. Because patients with CFRD are insulin-deficient, the standard medical treatment is exogenous insulin. Sulfonylureas enhance insulin secretion by acting on a specific islet beta cell receptor. No data are available about the outcome of sulfonylurea treatment vs. insulin treatment. In this retrospective study, data from 45 patients with CFRD were analyzed regarding their clinical outcome as it related to the treatment protocol. The duration of DM treatment was 7.6 +/- 4.6 years in the insulin-treated group and 3.5 +/- 2.0 years in the sulfonylurea group (n.s.). The age of CFRD diagnosis was significantly earlier in patients treated with insulin (n = 34) than in the patients treated with sulfonylurea (n = 11) (16.4 +/- 3.6 vs. 24.2 +/- 4.8 years, P < 0.001). No statistical differences were found between the two groups in the time of CF diagnosis, the most recent forced expired volume in 1 sec, forced vital capacity, Shwachman score, hemoglobin A(1C) levels, or weight for height index at the end of the study. Our data suggest that a subgroup of CFRD patients can be managed for a number of years with sulfonylurea, and that the clinical outcome was not different in this group compared with the insulin-treated patients.

Optimal tobramycin dosage in patients with cystic fibrosis--evidence for predictability based on previous drug monitoring.

A retrospective analysis of files of patients with cystic fibrosis and pulmonary exacerbations was performed to investigate whether an individual dosage of tobramycin once established by serum level determination allows a reliable prediction of the adequate dosage in a consecutive exacerbation. All patients hospitalized > or = 2 times between May 1997 and September 1998 with pulmonary exacerbation due to Pseudomonas aeruginosa infection susceptible to tobramycin were included. The initial dosage to tobramycin was 5 mg/kg body weight every 12 h followed by drug level determinations to establish the optimal dose. In a consecutive exacerbation the same dosage per kg body weight was used again and drug level determinations were repeated. Sixteen patients (six female = 38%) with a mean age of 24 years (median: 26 years, range: 9-33) were hospitalized for 49 pulmonary exacerbations (2-6 per patient, mean: 3, median: 2.5). During the first episode of tobramycin treatment in the study period all trough levels were < 2 microg/ml (median: 0.6) and the peak levels were 7.1-16.9 microg/ml (median: 11.9). In four patients the peak level was > 12 microg/ml. In 28 consecutive episodes the dosage of tobra myci n was chosen based on optimal results of previous drug level monitoring and in 27 instances (96%) the previously established optimal dose was confirmed. In five consecutive episodes the tobramycin dosage had been increased erroneously and this resulted in abnormally high peak levels in three cases. These findings suggest that a safe and therapeutic tobramycin dosage in an individual patient with cystic fibrosis is predictable based on a previously established optimal dosage.

Prospective evaluation of beta-cell function in insulin autoantibody-positive relatives of insulin-dependent diabetic patients.

During the preclinical period of insulin-dependent diabetes mellitus (IDDM), progression to clinical IDDM is characterized by declining beta-cell function. Although the presence of insulin autoantibodies (IAA) improves the ability of islet cell antibodies (ICA) to predict subsequent clinical IDDM, few studies have examined the risk of developing IDDM in subjects positive for IAA but negative for both ICA and antibodies to glutamic acid decarboxylase (64kA). To investigate this question, detailed beta-cell function tests (acute insulin response to glucose [AIRgluc] and slope of glucose potentiation) were performed on eight IAA-positive first-degree relatives of insulin-dependent diabetics. All eight subjects were negative for ICA, and seven were tested for 64kA and were negative. Five subjects were studied prospectively for 22.4 +/- 9.4 months, while three subjects had only initial studies. Initial beta-cell function tests were normal in each subject. AIRgluc was 122.2% +/- 19.0% of the expected normal response, while slope was 168.6% +/- 20.6% of expected normal response. beta-cell function remained normal and remarkably stable in the five subjects followed prospectively. AIRgluc did not significantly change from an initial value of 147.9% +/- 23.1% of expected to 153.2% +/- 22.4% (NS). The slope of glucose potentiation varied little from 165.5% +/- 39.4% initially to 159.5% +/- 27.3% (NS) at the most recent determination. We conclude that among nondiabetic first-degree relatives of IDDM subjects, the presence of IAA in the absence of ICA and 64kA is not usually associated with and therefore does not reliably predict beta-cell dysfunction or progressive deterioration in beta-cell function.

Quantitative analysis of islet glutamic acid decarboxylase p64 autoantibodies in insulin-dependent diabetes mellitus.

Autoantibodies against the beta-cell M(r) 64,000 protein (p64), recently identified as an isoform of glutamic acid decarboxylase (GAD), are prevalent in patients with insulin-dependent diabetes mellitus (IDDM). Dog islets were found to represent an abundant source of native p64 allowing the study of antigen-antibody interactions in IDDM. A quantitative, standardized assay for p64 antibodies based on dog islets was developed and evaluated. Utilizing dog and human islets the p64 antibodies were detected in 17/19 (89%) new onset 15-32-year-old patients, compared to 15/19 (79%) in a rat islet assay. ICA were detected in 15/19 (79%) patients and correlated with the presence of p64 antibodies (rs = 0.59, P < 0.004) but not with age at onset, sex, or C-peptide levels. Sensitivity therefore is improved with the dog islet p64 antibody assay which will allow future studies requiring native p64 antigen in larger quantities are possible based on our findings.

Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10.

Glutamic acid decarboxylase (GAD; glutamate decarboxylase, L-glutamate 1-carboxy-lyase, EC 4.1.1.15), which catalyzes formation of gamma-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, we describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different from that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows less than 65% identity to previously published, highly conserved GAD-1 brain sequences, which show greater than 96% deduced amino acid sequence homology among the three species. The function of this additional islet GAD isoform and its importance as an autoantigen in insulin-dependent diabetes remain to be determined.

Risk for developing type 1 (insulin-dependent) diabetes mellitus and the presence of islet 64K antibodies.

First-degree relatives of Type 1 (insulin-dependent) diabetic patients are at increased risk for developing clinical diabetes. The presence of islet cell or insulin autoantibodies further identifies relatives at greater risk, but not all immunologic-marker-positive relatives progress to disease. Beta-cell dysfunction, however, seems to be more prevalent than clinical Type 1 diabetes, since stable subclinical pancreatic Beta-cell dysfunction may occur. Antibodies against a Mr 64,000 (64K) islet Beta-cell protein, identified as glutamic acid decarboxylase, have been reported both at and several years prior to the clinical onset of Type 1 diabetes. We measured 64K antibodies in first-degree relatives with varying degrees of Beta-cell dysfunction and risk for subsequent Type 1 diabetes to determine whether 64K antibodies improve the predictive power of islet cell antibodies and/or insulin autoantibodies. In the Seattle Family Study first-degree relatives of Type 1 diabetic patients are followed prospectively using detailed Beta-cell function tests, insulin sensitivity, quantitative evaluation of islet cell antibodies and fluid phase assay insulin autoantibodies. 64K antibodies were measured using dog islets. Relatives were selected, based on Beta-cell function to represent individuals at high (n = 6) and low (n = 30) risk for subsequent Type 1 diabetes. The 30 low-risk individuals followed-up for 78 months, had stable Beta-cell function, and six (20%) were negative for all autoantibodies, ten (33%) were positive for insulin autoantibodies, 16 (53%) were islet cell antibody positive while six (20%) were positive for 64K antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmunity of diabetes.

Insulin-dependent diabetes mellitus is associated with a growing number of immune abnormalities. At the time of clinical onset, most patients developing the disease as children or young adults have autoantibodies reactive with islet beta cells. Current autoantibody markers for IDDM are not sufficient to predict the disease in the general population. Studies in first-degree relatives indicate the presence of a subclinical disease characterized by beta cell dysfunction, which may or may not progress to overt IDDM. Although IDDM is genetically linked to certain HLA-DQ class II molecules, it needs to be clarified whether these molecules determine the propensity to react to certain antigens, the failure to maintain tolerance, or the ability to produce disease-associated autoantibodies. Circumstantial evidence suggests that yet another gene outside the HLA complex on chromosome 6 is more important. The interaction with the environment needs to be clarified, and the etiologic role of viruses has not been substantiated. An underlying systemic autoimmune propensity may influence environmental insults and perpetuate islet beta cell destruction. Until these mechanisms are understood, clinicians should periodically check their patients with IDDM for other organ-specific as well as non--organ-specific autoimmune diseases. Our understanding of these phenomena is poor, which may explain why clinical trials with immunosuppressive agents have been of limited success. Further studies on the molecular biology of the immune response against islet beta cell-specific antigens are necessary for the development of both predictive tests and novel measures to prevent IDDM.

Metabolic state of the pancreas affects end-point titre in the islet cell antibody assay.

Quantification of islet cell antibodies is used increasingly to evaluate pre-clinical Type 1 (insulin-dependent) diabetes mellitus. If expression of the antigen(s) reacting with islet cell antibodies varies depending upon the functional state of the pancreatic islets, this may partly explain differences in assay sensitivity between laboratories. To address this question we altered Beta-cell function in Osborn-Mendel rats, by dietary manipulation prior to killing. Rats were fed chow (n = 7) or a high sucrose/high fat "cafeteria" diet (n = 8) or were fasted for 18 h (n = 6) until immediately prior to killing. Using frozen sections of these rat pancreata in the indirect immunofluorescent test for islet cell antibodies, we determined the end-point titre for 18 sera in which islet cell antibodies had been previously quantified in our standard human pancreas assay. These sera included ten positive sera and eight normal negative control sera. The four most strongly positive sera gave significantly higher end-point titres on "cafeteria" diet-fed pancreata and lower titres on fasted pancreata (p less than 0.04 to 0.002). None of non-diabetic control sera were positive on any substrate. These data suggest that islet antigen expression is increased when Beta-cell function is increased by dietary manipulation. Improved sensitivity in the islet cell antibody assay might be possible by altering Beta-cell function before or immediately after pancreas collection.

On the daily variation in the beta-receptor-adenylate cyclase-cAMP-phosphodiesterase system in rat forebrain.

In rat forebrain tissue of single rats beta-adrenoceptor density (Bmax) and affinity (Kd) were determined by saturation isotherms in receptor binding studies with the antagonist ligand (3H)-dihydroalprenolol at 8 different times of day in May. Rats were on a controlled 12L:12D photoperiod. In addition, the cAMP content, the formation of cAMP from ATP by the adenylate cyclase and the hydrolysis of the second messenger by the phosphodiesterase were determined at the same time points. No significant (ANOVA) daily variations were found in the total number of 3H-DHA binding sites (Bmax) nor in the affinity (Kd). In contrast, basal cAMP content as well as basal formation and hydrolysis of cAMP displayed significant rhythms. The peak value in cAMP was at the beginning of light. At that time the daily trough value in cAMP formation was found. Hydrolysis of cAMP by the phosphodiesterase displayed a 12-hr rhythm with trough values occurring at the early light and early dark period. The results demonstrate pronounced rhythmic changes in basal formation, content and hydrolysis of cAMP which are, however, not paralleled by changes in receptor number and/or affinity in the same tissue.