Microbiology of sputum from patients at cystic fibrosis centers in the United States.

During a phase III national collaborative study of aerosolized tobramycin (1 July 1995 through 30 September 1996), the microbiology of specimens from 595 patients at 69 cystic fibrosis (CF) centers was examined. Samples from three screening visits were processed in a single laboratory by means of standardized techniques for identification and susceptibility testing. From 1,753 pretreatment specimens, 5,128 pathogens were isolated (average, 2.9/specimen). Of the 3,936 Pseudomonas aeruginosa isolates, 56.7% were mucoid. The specimens of 125 patients (21.0%) yielded tobramycin-resistant P. aeruginosa (213 isolates); 61 (10.3%), Stenotrophomonas maltophilia; and 52 (8.7%), Alcaligenes xylosoxidans. Isolation of Burkholderia cepacia was an exclusion criterion. Only visit 3 sputum samples were cultured for gram-positive organisms and fungi (n = 465 patients); samples from 201 patients (43.2%) yielded Staphylococcus aureus (18.8% of isolates were oxacillin-resistant), and those from 114 (24.5%) yielded an Aspergillus species. Compared with the Cystic Fibrosis Foundation Patient Registry, the current study identified many more patients colonized with S. maltophilia, A. xylosoxidans, Aspergillus species, and oxacillin-resistant S. aureus, suggesting the utility of standardized processing of CF specimens.

Home nebulizer use among patients with cystic fibrosis.

OBJECTIVE: To describe current patterns of home nebulizer use among patients with cystic fibrosis. STUDY DESIGN: A population-based survey of home nebulizer practices among 227 patients with cystic fibrosis using nebulizers from 1993 to 1994 (Objective 1), and a prospective study of "typical" home use, including testing of performance and bacterial cultures in nebulizers after use, completed by 36 subjects (Objective 2). RESULTS: Objective 1: 85% of subjects reported using jet and 8% ultrasonic nebulizers (categories not mutually exclusive); 15% used unknown brands. Most jet nebulizers were disposable models, which were used for > 14 days by more than half the subjects. Mixing of medications in a single treatment (other than cromolyn and a bronchodilator) was reported by 28% of patients. Objective 2: no apparent deterioration in aerosol particle size or output rate of returned nebulizers compared with new units was observed. Staphylococcus aureus was cultured from 55% and Pseudomonas aeruginosa from 35% of returned nebulizers. Concordance between nebulizer and sputum cultures was poor. CONCLUSIONS: Although not generally tested for reusability, disposable nebulizers are generally used by patients for long periods. Medication mixing is common, although its effects on aerosol properties are unknown. Cystic fibrosis respiratory pathogens are frequently isolated from used nebulizers. Patient guidelines for home nebulizer use need to be established.

Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels in Xenopus eggs.

Previous experiments from our lab have suggested that the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is required for sperm-induced egg activation in Xenopus laevis. Here we measure the endogenous production of both Ins(1,4,5)P3 and PIP2 during the sperm-induced and ionomycin-induced calcium wave in the egg and find that both increase following fertilization. Ins(1,4,5)P3 increases 3.2-fold from an unfertilized egg level of 0.13 pmole per egg (0.29 microM) to a peak of 0.42 pmole per egg (0.93 microM) as the calcium wave reaches the antipode in the fertilized egg. This continuous production of Ins(1,4,5)P3 during the time that the Ca2+ wave is propagating across the egg suggests the involvement of Ins(1,4,5)P3 in wave propagation. This increase in Ins(1,4,5)P3 is smaller in ionomycin-activated eggs than in sperm-activated eggs, suggesting that the sperm-induced production of Ins(1,4,5)P3 involves a PIP2 hydrolysis pathway that is not simply raising intracellular Ca2+. While one might expect PIP2 levels to fall as a result of hydrolysis, we find that PIP2 actually increases 2-fold. The total lipid fraction in unfertilized egg exhibits 0.8 pmole PIP2 per egg and this increases to 1.5 pmole as the calcium wave reaches the antipode. The PIP2 concentration peaks 2 min after the completion of the calcium wave at 1.8 pmole per egg. The amount of PIP2 in the animal and vegetal hemispheres of the egg was also measured by cutting frozen eggs in half. The vegetal hemisphere contained twice the amount of PIP2 as the animal hemisphere but it also contained twice the amount of lipid. Thus, there was an equivalent amount of PIP2 normalized to lipid in each hemisphere. Isolated animal and vegetal hemisphere cortices exhibit similar PIP2 concentrations, suggesting that the 2-fold higher total PIP2 in the vegetal half is not due to a gradient of PIP2 in the plasma membrane, but rather implies that cytoplasmic organelle membranes also contain PIP2.

Highly polarized EGF receptor tyrosine kinase activity initiates egg activation in Xenopus.

Progesterone-matured Xenopus oocytes are arrested at second metaphase but resume meiosis following fertilization. To explore the role of tyrosine kinase activity and phosphatidylinositol turnover in this activation process, we caused oocytes to express three types of human epidermal growth factor receptor (EGF-R), which differ in their ability to stimulate these biochemical processes. Following mRNA injection we found that receptor expression was highly polarized, with most receptors located on the animal hemisphere. Occupancy of the wild-type EGF-R in progesterone-matured oocytes resulted in full egg activation as indicated by an activation potential, increased intracellular-free Ca2+ ([Ca2+]i), fertilization envelope liftoff, and cortical contraction. Fura-2 imaging showed that the wave of EGF-mediated Ca2+ release started in the animal hemisphere and progressed completely around the cell. These responses required receptor tyrosine kinase activity. Matured oocytes expressing the c'973 EGF-R, which possesses kinase activity but only weakly stimulates phosphatidylinositol turnover, responded differently to EGF addition. Cortical contraction and fertilization envelope liftoff appeared normal, but there was no activation potential. Significantly, [Ca2+]i was only slightly elevated and was topologically restricted to the regions expressing receptors. Our results suggest that some aspects of egg activation can occur through a tyrosine kinase pathway. However, phosphatidylinositol hydrolysis appears necessary for both amplification and propagation of signals generated locally by activated EGF-R.

The sperm-induced Ca2+ wave following fertilization of the Xenopus egg requires the production of Ins(1, 4, 5)P3.

We used fluorescence ratio imaging of fura-2 in the egg of Xenopus laevis to study the initiation and propagation of the wave of increased free Ca2+ that is normally triggered at fertilization. Naturally matured, jellied eggs were injected with fura-2 and ratio-imaged with fluorescence excitation at 350 and 385 nm while sperm were added. The [Ca2+]i rise normally begins as a small spot near the surface of the egg, remains fairly localized for 20-60 sec, and then spreads more rapidly across the egg at 7.5 +/- 0.05 microns/sec to reach the antipode about 5 min after fertilization. The [Ca2+]i wave velocity is slowed by increasing the concentration of fura-2 in the cytoplasm to 100 microM, and 250 microM fura-2 blocks wave propagation. The peak [Ca2+]i in a sperm-activated wave is 2.2 +/- 0.1 microM and [Ca2+]i returns to preactivation levels within 21 +/- 0.7 min after fertilization. We further studied the mechanism by which sperm trigger the Ca2+ wave by injecting substances that interfere with Ins(1,4,5)P3-induced Ca2+ release or Ca(2+)-induced Ca2+ release (CICR). Heparin (3 kDa) inhibits sperm-induced egg activation in a manner that is linearly proportional to its cytoplasmic concentration. At 130 microM (390 micrograms/ml), sperm-induced activation is completely blocked and at 75 microM (225 micrograms/ml) activation of half of the eggs is inhibited. All eggs injected with 130 microM heparin are polyspermic as verified using Hoechst dye to label nuclear DNA. Imaging eggs injected with 75 microM heparin revealed multiple, transient "spots" of increased [Ca2+]i that failed to spread across the egg. Injection of a monoclonal antibody to PIP2 (0.2 microM), kt3g, blocked sperm-induced egg activation in 73% of the 30 eggs injected, suggesting that activation requires the hydrolysis of PIP2 in the membranes of the egg rather than simply the introduction of Ins(1,4,5)P3 from the sperm. This sperm-induced egg activation is not blocked by either of two CICR inhibitors, procaine (10 mM) or ruthenium red (30 microM), and egg activation is not triggered by either of two stimulators of CICR, caffeine (10 mM) or ryanodine (50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)