Plasma and serum lactoferrin levels in cystic fibrosis. Relationship with the presence of cystic fibrosis protein

Plasma lactoferrin concentrations were measured in blood of cystic fibrosis patients, heterozygotes and controls using a specific and sensitive enzyme immunoassay. 67 plasmas were studied (26 controls, 23 heterozygotes, 18 cystic fibrosis patients) and the results showed a statistically significant increase (p less than 0.05) of the level of plasma lactoferrin in cystic fibrosis patients (265 +/- 224 micrograms/l) compared to controls (168 +/- 100 micrograms/l) and heterozygotes (150 +/- 72 micrograms/l). Since it is well established that plasma lactoferrin level could be influenced by the number of neutrophils, a second set of experiments was performed on 20 cystic fibrosis patients on whom leukocyte counts were also made. When the 15 plasmas with normal neutrophils (in the range 2 to 6 giga/l) were considered, the mean lactoferrin level was 318 +/- 116 micrograms/l, still far above the normal values. For serum, a similar significant increase of lactoferrin concentration was observed in 33 cystic fibrosis patients (610 +/- 551 micrograms/l) compared to the values observed for 25 controls (237 +/- 155 micrograms/l) and 37 heterozygotes (272 +/- 231 micrograms/l). Cystic fibrosis protein (CFP) was identified in the same sera by isoelectric focusing and the intensity of the band was closely related to the increase of lactoferrin concentration in cystic fibrosis patients. In contrast, no difference in serum lactoferrin concentrations was observed between heterozygotes with or without CFP, indicating that the increased CFP concentration cannot be due only to altered granulocyte function.     

Plasma lactoferrin levels in pregnancy and cystic fibrosis

Plasma lactoferrin levels were determined by radioimmunoassay for the different weeks of normal pregnancy, in normal healthy adults and in children with and without cystic fibrosis.The lactoferrin levels were higher in pregnancy than in both male and female normal adults and showed a slight progressive increase up to week 29 and thereafter remained high.Five out of seven children with cystic fibrosis had markedly raised plasma lactoferrin levels from six to 16 times higher than the mean of a control group of children.     

Relative ion permeability of normal and cystic fibrosis nasal epithelium

The raised transepithelial electric potential difference (PD) across respiratory epithelia in cystic fibrosis (CF) has suggested an abnormality in ion permeation. We characterized this abnormality further by measuring in the nasal epithelia of CF and normal subjects the concentration-PD relationship for amiloride, an inhibitor of cell Na+ permeability, and PD responses to superfusion with solutions of different composition. Amiloride was more efficacious in the CF subjects but the ED50 was not different from that of normals (approximately 2 X 10(-6) M). Na+ replacement by choline induced effects similar to those of amiloride, i.e. a greater depolarization in CF subjects. A 10-fold increase in the K+ concentration of the perfusate induced a small (less than 10 mV) depolarization in both subject populations. When Cl- in the perfusate was replaced by gluconate or SO2-(4) the nasal PD of normal subjects hyperpolarized (lumen became more negative) by approximately 35 mV. A significantly smaller response (less than 17 mV) was induced in CF homozygotes but not in heterozygotes (38 mV). The smaller response of CF subjects appears to reflect an absolute decrease in luminal surface Cl- permeability because pretreatment with amiloride did not increase the response to Cl- free solution (7 mV). Accordingly, three abnormalities (decreased Cl- permeability, raised PD, greater amiloride efficacy) have been identified in CF respiratory epithelia. Whereas "excessive" active Na+ transport can account for these abnormalities and the dessication of airway surface liquid, it is possible that a lower lumenal cell membrane Cl- permeability and inhibition of a potential path of Cl- secretion can also explain the observations.     

Cyclic adenosine monophosphate-dependent kinase in cystic fibrosis tracheal epithelium.

Cl-impermeability in cystic fibrosis (CF) tracheal epithelium derives from a deficiency in the beta-adrenergic regulation of apical membrane Cl- channels. To test the possibility that cAMP-dependent kinase is the cause of this deficiency, we assayed this kinase in soluble fractions from cultured airway epithelial cells, including CF human tracheal epithelial cells. Varying levels of cAMP were used in these assays to derive both a Vmax and apparent dissociation constant (Kd) for the enzymes in soluble extracts. The cAMP-dependent protein kinase from CF human tracheal epithelial cells has essentially the same Vmax and apparent Kd as non-CF human, bovine, and dog tracheal epithelial cells. Thus, the total activity of the cAMP-dependent kinases and their overall responsiveness to cAMP are unchanged in CF.     

Serum sulfate levels in patients with cystic fibrosis

Patients with cystic fibrosis (CF) secrete copious amounts of mucous material which is viscous, tends to accumulate in the respiratory tract and contains larger than normal amounts of sulfate. The present investigation was designed to measure sulfate levels in the serum of patients with cystic fibrosis by ion chromatography of protein-free serum aliquots. The level of inorganic sulfate in the serum of non-cystic fibrosis pediatric patients averaged 0.29 ± 0.03 mmol/l while patients suffering from cystic fibrosis had an average serum sulfate value of 0.27 ± 0.03 mmol/l which was not significantly different from controls. No differences were observed in serum sulfate levels among males and females of either group of patients. There was a tendency for serum sulfate levels to decrease with age, but there was no statistically significant difference in serum sulfate levels between cystic fibrosis patients and normals as a function of age. These findings indicate that the highly sulfated mucoid materials secreted by cystic fibrosis patients are not reflected in abnormal serum sulfate levels.     

Polyamine alterations in blood of male homozygotes and heterozygotes for cystic fibrosis.

The polyamines, spermidine and spermine, have been measured in whole blood extracts from control volunteers, patients with cystic fibrosis, and obligate cystic fibrosis heterozygotes. Male homo- and heterozygotes for cystic fibrosis exhibit a consistent and significant decrease in blood spermine resulting in an elevated spermidine/spermine ratio when compared to control males. In contrast, control females exhibited reduced spermidine and spermine levels as compared to control males. Blood from female cystic fibrosis homo- and heterozygotes showed similar results. The sex difference is probably due to fluctiations of blood polyamines during the menstrual cycle. Abnormal polyamine levels are the first observation of an alteration of a low molecular weight metabolite characteristic of both male homo- and heterozygotes for cystic fibrosis.     

Endocytosis and intracellular protein degradation in cystic fibrosis fibroblasts

Normal rates of pinocytosis of [3H]sucrose were measured in cystic fibrosis fibroblasts, and were not affected by the addition of cystic fibrosis serum. Bulk protein degradation (a significant proportion of which occurs intralysosomally following autophagy) and its regulation by growth state was apparently identical in normal and cystic fibrosis cultures.     

Increased soluble CD40 ligand levels in cystic fibrosis

Summary. Chronic inflammation represents a key pathogeneric event in the progression of lung disease in cystic fibrosis (CF). To identify novel mechanisms of the inflammatory reaction in CF and analyze its relation with coagulative activation, we carried‐out a cross‐sectional study to evaluate circulating levels of the inflammatory mediators soluble (s) CD40L, C‐reactive protein (CRP), interleukin (IL)‐1β, the coagulation markers activated factor VII (FVIIa) and prothrombin fragment (F) 1+2, as well as urinary 11‐dehydro‐thromboxane (TX)B₂, an index of in vivo platelet activation, in 34 CF patients and 34 matched healthy subjects. We observed that CF patients displayed significantly increased circulating levels of sCD40L compared to controls [2.8 (0.4–15.6) vs 1.1 (0.2–2.7) ng mL^?1 ,P = 0.0003]. sCD40L levels inversely correlated with forced expiratory volume at 1 second (FEV₁) (ρ = ?0.788, P = 0.0001), whereas it directly correlated with CRP and IL‐1β levels (ρ = 0.621, P = 0.0004; and ρ = 0.745, P = 0.0001, respectively), which were also elevated in CF patients. CF patients had also enhanced levels of FVIIa and F1+2 compared to controls [39.2 (22.6–69.8) vs 22.3 (16.2–32.4) mU mL^?1, P = 0.0001; 0.60 (0.30–1.80) vs 0.17 (0.10–0.40) nmol L^?1, P = 0.0001, respectively]. A direct correlation was observed between sCD40L and both plasma FVIIa (ρ = 0.691, P = 0.0001) and F1+2 (ρ = 0.545, P = 0.0017) as well as between sCD40L and urinary 11‐dehydro‐TXB₂ (ρ = 0.433, P = 0.0129). Our findings suggest that in CF patients, sCD40L could represent a biochemical link between the inflammatory state, and endothelial damage and coagulative activation, leading to progressive impairment of pulmonary function.     

In vivo physiologic comparison of two ventilators used for domiciliary ventilation in children with cystic fibrosis.

OBJECTIVE: Home noninvasive mechanical ventilation (NIMV) is used with increasing frequency for the treatment of patients with respiratory failure caused by cystic fibrosis, yet the optimal mode of ventilation in such children is unknown. We compared the physiologic short-term effects of two ventilators with different modes (one pressure support and the other assist control/volume-targeted [AC/VT]) commonly used for domiciliary ventilation. DESIGN: Prospective, randomized, crossover comparison of two ventilators with different modes. SETTING: Tertiary pediatric university hospital. PATIENTS: Eight children with cystic fibrosis (age, 11-17 yrs) and chronic respiratory failure (pH 7.4 +/- 0.0; PaO2, 57.5 +/- 7.5 torr; PaCO2, 46.1 +/- 2.5 torr), naive to NIMV. INTERVENTIONS: Two 20-min runs of pressure support and AC/VT ventilation were performed in random order, each run being preceded and followed by 20 mins of spontaneous breathing. MEASUREMENTS: Flow and airway pressure and esophageal and gastric pressures were measured to calculate esophageal (PTPes) and diaphragmatic pressure-time product (PTPdi) and the work of breathing. RESULTS: The two NIMV sessions significantly improved blood gas variables and increased tidal volume with no change in respiratory rate. Indexes of respiratory effort decreased significantly during the two modes of NIMV compared with spontaneous breathing, with PTPdi/min decreasing from 497.8 +/- 115.4 cm H2O x sec x min(-1) during spontaneous breathing to 127.8 +/- 98.3 cm H2O x sec x min(-1) and 184.3 +/- 79.8 cm H2O x sec x min(-1), during AC/VT and pressure support, respectively (p <.0001), and the work of breathing decreasing from 1.83 +/- 0.12 J.L-1 during spontaneous breathing to 0.48 +/- 0.32 J.L-1 and 0.75 +/- 0.30 J.L-1, during AC/VT and pressure support, respectively (p <.0001). In addition, the effect of AC/VT ventilation was significantly superior to pressure support judged by PTPes and the work of breathing, but this result was explained by three patients who adapted extremely well to the AC/VT ventilation, with the disappearance of ventilator triggering, in effect adopting a controlled mode. There was a correlation between the improvement in PTPdi/min or the work of breathing and patient's subjective impression of comfort during the AC/VT ventilation. CONCLUSIONS: In awake, stable children with cystic fibrosis, both AC/VT and pressure support unloaded the respiratory muscles. The disappearance of ventilator triggering occurred in a subgroup of patients during AC/VT ventilation, and this explained the good tolerance and the superiority of this mode in the present study.     

Increased circulating levels of plasma ATP in cystic fibrosis patients

Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP‐binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin‐luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single‐blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0·01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the ΔF508 genotype had a 54% (n=33, P<0·01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0·4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease.     

Electrolytes and norepinephrine levels in blood of patients with cystic fibrosis.

Young adults with cystic fibrosis in good to excellent condition have reduced plasma sodium and chloride and elevated plasma potassium compared to a group of healthy young adult control subjects. Blood pressure was also lower in the patients with cystic fibrosis. However, plasma norepinephrine and dopamine-beta-hydroxylase and their response to standing and isometric hand grip were normal in the cystic fibrosis patients.     

Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Recombinant adenoviruses have shown promise as vectors for transfer of CF transmembrane conductance regulator cDNA to airway epithelia and correction of the Cl- transport defect. However, because adenovirus-mediated gene transfer is transient, use of adenovirus as a vector for treatment of CF would require repeated administration. Therefore, we evaluated repeat administration of an adenovirus vector to the nasal epithelium of patients with CF with five escalating doses of up to 10(10) infectious units. There were no detectable adverse affects. All subjects were initially seropositive but developed additional humoral immune responses. The vector partially corrected the defect in airway epithelial Cl- transport in some subjects, although there was variability between subjects and there was less correction with subsequent administration, perhaps because the immune response limited gene transfer. Future work must focus on vectors with increased efficiency and with the ability to evade host defenses.     

In vivo antitumor activity and induction of insulin-like growth factor-1-resistant apoptosis by SC-alphaalphadelta9

We previously showed that SC-alphaalphadelta9 (4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid) is a novel antiphosphatase agent that selectively inhibits the growth of transformed cells in culture and affects elements of insulin-like growth factor-1 (IGF-1) signaling. We now show that SC-alphaalphadelta9 induces IGF-1-resistant apoptosis and kills tumor cells in vivo. In cultured murine 32D cells, SC-alphaalphadelta9 induced concentration-dependent apoptosis that was blocked by ectopic Bcl-2 expression. No apoptosis was detected in 32D cells treated with the congener SC-alpha109, which lacks the ability to disrupt IGF-1 signaling. After interleukin-3 withdrawal or etoposide treatment, exogenous IGF-1 prevented apoptosis and elevated levels of Cdc2, a biochemical indicator of a functional IGF-1 receptor pathway. In contrast, exogenous IGF-1 did not prevent apoptosis or loss of Cdc2 expression caused by SC-alphaalphadelta9. Furthermore, IGF-1 receptor overexpression failed to protect cells against SC-alphaalphadelta9-induced apoptosis. Kinetic analyses demonstrated that Cdc2 down-regulation after SC-alphaalphadelta9 treatment preceded both apoptosis and loss of the IGF-1 receptor, indicating that loss of Cdc2 was a direct effect of SC-alphaalphadelta9 treatment and not secondary to cell death. IGF-1 receptor autophosphorylation studies indicated that SC-alphaalphadelta9 did not interact directly with the IGF-1 receptor nor bind to the growth factor itself, suggesting a site of action distal to the IGF-1 receptor. In the SCCVII murine tumor model, a single i.p. injection of SC-alphaalphadelta9 caused a dose-dependent decrease in clonogenic cell survival. The IC(50) of SC-alphaalphadelta9 was 35 mg/kg, comparable to 25 mg/kg carboplatin. The ability to induce IGF-1-resistant apoptosis distinguishes SC-alphaalphadelta9 from other apoptosis-inducing agents and suggests compounds of this class deserve further study as potential anticancer agents.     

Effect of ibuprofen on neutrophil migration in vivo in cystic fibrosis and healthy subjects

Long-term treatment with ibuprofen twice daily, at doses that achieve peak plasma concentration (Cmax) >50 microg/ml, slows progression of lung disease in patients with cystic fibrosis (CF). Previous data suggest that Cmax >50 microg/ml is associated with a reduction in neutrophil (PMN) migration into the lung and that lower concentrations are associated with an increase in PMN migration. To estimate the threshold concentration at which ibuprofen is associated with a decrease in PMN migration in vivo, we measured the PMN content of oral mucosal washes in 35 healthy (age 19-40 years) and 16 CF (age 18-32 years) subjects who took ibuprofen twice daily for 10 days in doses that achieved Cmax 8 to 90 microg/ml. Cmax >50 microg/ml was associated with a 31 +/- 7% (mean +/- S.E.M.) reduction in PMNs in CF (n = 11, p < 0.001) and 25 +/- 6% reduction in PMNs in healthy subjects (n = 16, p < 0.001). Increasing concentrations above 50 microg/ml was not associated with a greater decrease in PMNs. The reduction in PMN migration was consistently present 12 h after a dose, but not after 24 h. Cmax <50 microg/ml was associated with an increase in PMNs of approximately 40%. These results suggest that Cmax >50 microg/ml and twice daily dosing of ibuprofen are required to decrease PMN migration, and reinforce the current recommendation that pharmacokinetics should be performed in CF patients prescribed ibuprofen.     

In vitro alterations of tracheal epithelium of hamsters by carcinogens in organ culture

In vitro organ culture of the hamster's trachea was improved and applied to a carcinogenesis research. The rotary culture enabled explants of tracheal epithelium to survive more than 8 weeks. The study was composed of 2 kinds of culture; untreated and treated with carcinogens. In the untreated culture, Eagle MEM medium had the same culture effect as RPMI 1640 medium. With prolongation of culture time (particularly longer than 5 weeks), irreversible degenerative changes appeared in epithelial cells. Culture for 4 weeks was usually thought to be appropriate for experimental research. In the treated culture, the effect of benzo-(a) pyrene (B(a)P) and B(a)P + cigarette smoking condensate-neutral fraction (CSC-NF) on tracheal epithelium was investigated with light and electron microscopies (TEM and SEM) and autoradiography. Atypical hyperplasia with or without lesions suggesting carcinoma in situ was induced by B(a)P + CSC-NF more evidently and frequently than by B(a)P alone. The present findings corroborated the cocarcinogenetic effect of CSC-NF.