Phase II studies of nebulised Arikace in CF patients with Pseudomonas aeruginosa infection.

RATIONALE: Arikace is a liposomal amikacin preparation for aerosol delivery with potent Pseudomonas aeruginosa killing and prolonged lung deposition. OBJECTIVES: To examine the safety and efficacy of 28 days of once-daily Arikace in cystic fibrosis (CF) patients chronically infected with P aeruginosa. METHODS: 105 subjects were evaluated in double-blind, placebo-controlled studies. Subjects were randomised to once-daily Arikace (70, 140, 280 and 560 mg; n=7, 5, 21 and 36 subjects) or placebo (n=36) for 28 days. Primary outcomes included safety and tolerability. Secondary outcomes included lung function (forced expiratory volume at one second (FEV1)), P aeruginosa density in sputum, and the Cystic Fibrosis Quality of Life Questionnaire-Revised (CFQ-R). RESULTS: The adverse event profile was similar among Arikace and placebo subjects. The relative change in FEV1 was higher in the 560 mg dose group at day 28 (p=0.033) and at day 56 (28 days post-treatment, 0.093L±0.203 vs -0.032L±0.119; p=0.003) versus placebo. Sputum P aeruginosa density decreased >1 log in the 560 mg group versus placebo (days 14, 28 and 35; p=0.021). The Respiratory Domain of the CFQ-R increased by the Minimal Clinically Important Difference (MCID) in 67% of Arikace subjects (560 mg) versus 36% of placebo (p=0.006), and correlated with FEV1 improvements at days 14, 28 and 42 (p<0.05). An open-label extension (560 mg Arikace) for 28 days followed by 56 days off over six cycles confirmed durable improvements in lung function and sputum P aeruginosa density (n=49). CONCLUSIONS: Once-daily Arikace demonstrated acute tolerability, safety, biologic activity and efficacy in patients with CF with P aeruginosa infection.

Interaction of prenatal exposure to cigarettes and MAOA genotype in pathways to youth antisocial behavior.

Genetic susceptibility to antisocial behavior may increase fetal sensitivity to prenatal exposure to cigarette smoke. Testing putative gene x exposure mechanisms requires precise measurement of exposure and outcomes. We tested whether a functional polymorphism in the gene encoding the enzyme monoamine oxidase A (MAOA) interacts with exposure to predict pathways to adolescent antisocial behavior. We assessed both clinical and information-processing outcomes. One hundred seventy-six adolescents and their mothers participated in a follow-up of a pregnancy cohort with well-characterized exposure. A sex-specific pattern of gene x exposure interaction was detected. Exposed boys with the low-activity MAOA 5' uVNTR (untranslated region variable number of tandem repeats) genotype were at increased risk for conduct disorder (CD) symptoms. In contrast, exposed girls with the high-activity MAOA uVNTR genotype were at increased risk for both CD symptoms and hostile attribution bias on a face-processing task. There was no evidence of a gene-environment correlation (rGE). Findings suggest that the MAOA uVNTR genotype, prenatal exposure to cigarettes and sex interact to predict antisocial behavior and related information-processing patterns. Future research to replicate and extend these findings should focus on elucidating how gene x exposure interactions may shape behavior through associated changes in brain function.

Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa.

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation.

In previous work (E. E. Smith, D. G. Buckley, Z. Wu, C. Saenphimmachack, L. R. Hoffman, D. A. D'Argenio, S. I. Miller, B. W. Ramsey, D. P. Speert, S. M. Moskowitz, J. L. Burns, R. Kaul, and M. V. Olson, Proc. Natl. Acad. Sci. USA 103:8487-8492, 2006) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.

KB001-A, a novel anti-inflammatory, found to be safe and well-tolerated in cystic fibrosis patients infected with Pseudomonas aeruginosa.

BACKGROUND: Chronic Pseudomonas aeruginosa (Pa) airways infection, exuberant local inflammation, and progressive lung function loss are hallmarks of cystic fibrosis (CF). KB001-A is an anti-PcrV PEGylated monoclonal antibody fragment to the Type III secretion system of Pa. This 16-week study evaluated KB001-A associated effect on time-to-need for antibiotics for worsening respiratory signs and symptoms, as well as safety, and treatment-associated changes in symptom scores, inflammatory markers, and spirometry. METHODS: This was a randomized, double-blind, placebo-controlled, repeat-dose study in CF subjects with Pa. Intravenous 10mg/kg KB001-A or placebo infusions were administered at baseline and weeks 2, 4, 8, and 16, with a 4-week follow-up. Sputum inflammatory markers were assessed in a sub-study. Time-to-need for antibiotics was compared between groups by Kaplan Meier analysis and Cox proportional hazards modeling adjusting for randomization strata. RESULTS: Of 182 subjects, 169 received at least one infusion of KB001-A (n=83) or placebo (n=86). KB001-A was generally safe and well-tolerated as compared to placebo, with no significant emergent adverse effects other than one serious adverse event of elevated hepatic enzymes of unclear etiology. Time to need for antibiotics did not differ between groups (HR: 1.00; 95% CI: 0.69, 1.45, p=0.995). A 3.2 increase in ppFEV1 from placebo favoring KB001-A was observed at week 16 (95% CI: 1.12, 5.30, p=0.003). Mean changes from baseline in log10 sputum neutrophil elastase (NE) had a non-significant decrease (-0.27, 95% CI: -0.58,0.04, p=0.084) while IL-8 concentrations at week 16 were significantly lower (-0.27, 95% CI: -0.55,0.00, p=0.048) among KB001-A subjects (n=16) relative to placebo (n=13). CONCLUSIONS: KB001-A was safe and well-tolerated and associated with a modest FEV1 benefit and reduction in select sputum inflammatory markers (IL-8). KB001-A was not associated with an increased time to need for antibiotics. The lack of efficacy seen with KB001-A may be due, in part, to the low levels of the type III secretion proteins previously reported in sputum of CF patients chronically infected with Pa.

Covariates of tooth-brushing frequency in low-income African Americans from grades 5 to 8.

PURPOSE: The purpose of this study was to examine tooth-brushing frequency in 575 urban and nearby suburban African American children as part of a comprehensive risk-reduction study for students at high risk for violence, drugs, school delinquency, and unsafe sexual behaviors to determine which covariates predicted tooth-brushing frequency. METHODS: Students were surveyed 5 times, from the beginning of grade 5 and the end of each year through grade 8, and parents were surveyed at the beginning of grade 5. Peer influence, importance of being liked, self-esteem, attitudes towards tooth-brushing, oral health knowledge, self-efficacy, parental attitudes, and other covariates were examined for the ability to predict self-reporting of tooth-brushing frequency. RESULTS: In the fifth grade, peer influence, the importance of being liked, and physical self-esteem were the significant predictors, and peer influence continued to predict tooth-brushing in the eighth grade. Oral health knowledge and parental influence were not significant. CONCLUSION: Peer influence is an important factor in tooth-brushing behavior in metropolitan African American preadolescent children.

Open pulmonary biopsy. Nineteen-year experience with 416 consecutive operations.

Four hundred and sixteen open pulmonary biopsies through limited thoracotomies are reported. Tissue sufficient for diagnosis was obtained in all cases. Case selection, operative technique, spectrum of diagnoses, complications, and comparisons with other techniques are defined. Diagnoses by category were as follows: occupational, 105 patients (25 percent); neoplastic disease, 80 patients (19 percent); specific histologic diagnosis, (ie, sarcoidosis), 70 patients (17 percent); specific infection, 23 patients (6 percent); vascular diagnosis, 16 patients (4 percent); and nonspecific pulmonary disease, 122 patients (29 percent). Pneumothorax, minor in most cases, was the most common complication. It occurred in 97 (23 percent) of the patients, but only 24 (6 percent) required the placement of a chest tube. Pleural effusion occurred in 106 patients (25 percent) and was minor. Hemothorax occurred in two (0.5 percent) and superficial wound infection in three (0.7 percent). Overall mortality was 4.5 percent (19 patients). Only two deaths (0.4 percent) were related to the procedure. Open pulmonary biopsy remains our diagnostic method of choice in diffuse lung disease of undetermined etiology.

Fifteen years of experience with bacterial meningitis.

BACKGROUND: Introduction of Haemophilus influenzae type b (Hib) vaccines has dramatically altered the epidemiology of bacterial meningitis in children. The goal of this study was to describe these changes in a pediatric teaching hospital. METHODS: Patient charts at Children's Hospital and Regional Medical Center, Seattle, were identified by diagnosis codes and reviewed retrospectively. The 1981 to 1995 time period was chosen to incorporate three distinct 5-year periods: before the use of unconjugated Hib vaccine; between the unconjugated and conjugate vaccines; and after the conjugate vaccines were available for routine immunization of infants. RESULTS: Bacterial meningitis was identified in 806 cases. In 13 premature infant cases Escherichia coli was most frequently isolated (6 cases). Group B Streptococcus, E. coli and Listeria monocytogenes were the most common pathogens in 87 neonatal cases. The most common pathogens in 706 cases of childhood meningitis were H. influenzae, Streptococcus pneumoniae and Neisseria meningitidis. H. influenzae was the most common pathogen in the first two time periods (73 and 69% of childhood cases, respectively), but not so in the third period (16%). CONCLUSIONS: A changing pattern in childhood meningitis was observed during the study period. H. influenzae cases dramatically declined, altering the relative proportions of other pathogens, S. pneumoniae and N. meningitidis. However, the number of cases caused by these latter pathogens remained steady.

A temperature-jump device for time-resolved cryo-transmission electron microscopy.

We describe a temperature-jump device that permits time-resolved studies of thin cryo-transmission electron microscopy specimens. The specimen is rapidly heated to induce a change in microstructure just prior to cryo-fixation. The apparatus consists of a xenon arc lamp equipped with a shutter controlled by timing circuitry, used in conjunction with an environmental specimen preparation chamber. The specimen is heated by exposure to focused light from the lamp, and then plunged into cryogen. Using a thermocouple constructed from an electron microscope grid, we show that temperature jumps of 30-60 K are achieved with exposure times of 150-450 milliseconds. Micrographs of dimyristoyl phosphatidylcholine (DMPC) vesicles and n-docosane films, subjected to these exposures, show that the specimens are still at least 20-30 K above their initial temperature when they contact the cryogen. This method could be applied to a variety of biological and chemical systems which undergo structural changes activated by a rise in temperature.

Comparison of two commercial systems (Vitek and MicroScan-WalkAway) for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Antimicrobial susceptibility testing of cystic fibrosis (CF) isolates of Pseudomonas aeruginosa is difficult because the organisms are often mucoid and slow-growing. This study of 498 CF strains examined the correlation of results derived from two commonly used commercial systems (Vitek, MicroScan-WalkAway) with a reference method for 10 antimicrobials. Correlation to reference results was unacceptably low for all agents and both commercial systems had a high rate of very major (false-susceptible) errors. Although mucoid strains produced a 4.8% greater intermethod error, it was not markedly different than non-mucoid strains for the Vitek System. Overall, these tested commercial systems performed poorly for CF isolates in contrast to earlier reported, high correlations with the reference methods (broth microdilution frozen panels and agar dilution) of the National Committee for Clinical Laboratory Standards, the standardized disk diffusion test, and the Etest (AB BIODISK, Solna, Sweden).

Early pulmonary infection, inflammation, and clinical outcomes in infants with cystic fibrosis.

A thorough understanding of the early natural history of cystic fibrosis (CF) lung disease is critical for the development of effective interventions in the youngest patients. We assessed the evolution of pulmonary infection, inflammation, and clinical course among 40 infants over a 2-year period through annual bronchoalveolar lavage (BAL) for culture and measurements of pro- and anti-inflammatory cytokines, semiannual infant pulmonary function testing, and quarterly clinical evaluations. Both the prevalence of CF pathogens and their density in BAL fluid increased with age. Infants had neutrophilic lower airway inflammation and elevated IL-8 concentrations independent of whether CF pathogens were recovered. Total leukocyte and neutrophil densities and IL-8 concentrations increased with density of CF pathogens in BAL fluid, whether the isolated organism was P. aeruginosa or another pathogen. IL-10 concentrations were similar in CF subjects and non-CF historical controls. Infants generally had suboptimal growth (low weight and height percentiles) and obstructive lung disease (decreased expiratory flows and air trapping). Subjects from whom CF pathogens were isolated at > 10(5) cfu/mL had the worst air trapping and lowest Brasfield chest X-ray scores. Our findings provide a foundation for future studies of early intervention in CF lung disease, including antimicrobial and anti-inflammatory therapy.

Preparation and characterization of a reconstituted stratum corneum film as a model membrane for skin transport.

A zwitterionic surfactant, 6-eicosyldimethyl ammoniohexanoate (C20AH), completely disaggregates stratum corneum into individual cells. The cells can be cast into a film of reconstituted stratum corneum (RSC). Such films prepared from pig and human skin mimic intact human stratum corneum in microscopic, mechanical, and barrier evaluations. The films are useful as model membranes for skin transport experiments.

Changes in visceral yolk sac ultrastructure after exposure of rat embryos to selected teratogens in vitro.

It has been determined that a number of teratogens alter the osmotic environment around the rat embryo, an effect that is associated with abnormal fluid accumulation (and ultimately abnormality) in the embryo. At least one of these teratogens, trypan blue, changes lysosomal structure in the visceral yolk sac (VYS), an extra-embryonic membrane that envelops the extra-embryonic fluid compartment. The osmotic and ultrastructural effects are comparable in the in vivo and in vitro rat embryo. In the present study, the effects of other osmotic teratogens on VYS ultrastructure were investigated in rat whole embryo culture. Leupeptin (10 mug/ml) and E-64 (10 mug/ml) both caused a marked increase in the size of VYS lysosomes. Both chemicals inhibit cysteine proteinases, which are abundant in lysosomes. Suramin (750 mug/ml), an inhibitor of a number of lysosomal hydrolases, caused vacuolization of large areas of VYS cells. Ethylenethiourea (120 mug/ml) produced no marked ultrastructural changes, although the endocytotic apparatus of VYS cells appeared to have increased electron density, an effect that was also observed after treatment with the other teratogens. These results indicate that teratogens which alter embryonic osmotic balance also affect structures involved in endocytosis or lysosomal degradation of material by VYS cells.

Establishment of immortalized alveolar type II epithelial cell lines from adult rats.

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of bacterial resistance.

Antibiotic resistance is a growing problem in clinical pediatrics. Many of the agents traditionally used to treat pediatric pathogens are becoming less effective because of increasing bacterial resistance. In addition, many more children are immunocompromised because of primary or acquired immunodeficiencies and because of advances in cancer chemotherapy and transplantation. These children are being admitted to hospitals where they may be exposed to multiply resistant nosocomial pathogens. An improved understanding of the mechanisms of antibiotic resistance and the development of treatment strategies to prevent the emergence of resistance will be increasingly required in pediatrics.

Pediatric urinary tract infection. Diagnosis, classification, and significance.

The initial task is to establish the diagnosis of a urinary tract infection. The clinical setting, method of specimen collection, bacterial colony count, and species are all important considerations. Next, the infection is classified as complicated or uncomplicated. Complicated infections require hospitalization and parenteral antibiotic therapy. Appropriate imaging studies are imperative to determine whether urologic intervention is necessary. All children with well-documented urinary tract infections deserve diagnostic evaluation, regardless of sex or presence of systemic symptoms.

Decalin-induced nephrotoxicity: light and electron microscopic examination of the effects of oral dosing on the development of kidney lesions in the rat.

Male and female Fischer 344 rats were given decalin by oral gavage for 5 or 12 consecutive days in order to determine whether oral dosing would result in light microscopically evident renal effects that were comparable to those that have been observed after inhalation exposure. Decalin (in corn oil vehicle) was administered at doses of 0, 0.1, 0.5, 1.0 or 2.0 g/kg body weight to male rats, and 0, 1.0, 1.5, 1.75 or 2.0 g/kg to female rats. Biopsies of the kidneys of selected control and high-dose male rats were taken for examination by electron microscopy. Sections of kidneys from all control and treated rats were examined by light microscopy. The kidneys of all male control rats contained minimal levels of hyaline droplets within the cytoplasm of proximal convoluted tubule (PCT) epithelial cells. Decalin-induced alterations in the kidneys of male rats included an exacerbation of the hyaline droplet/globule levels found in controls and the formation of granular casts in the outer zone of the renal medulla. The exacerbated formation of hyaline droplets was characterized light microscopically by a marked dose-related increase in the number and size of individual droplets/globules and ultrastructurally by a marked increase in the size range of, and the presence of crystalline inclusions in, the PCT epithelial cell phagolysosomal populations. No other ultrastructural alterations occurred that differentiated treated male rats from control males. The formation of granular casts was dose and time related, occurring in 60% of male rats given 0.5 g decalin/kg for 12 days and in 100% of those given 1.0 g decalin/kg for 12 days. Light microscopy revealed no differences between the kidneys of control and decalin-treated female rats, and no hyaline droplets or granular casts were observed in the kidneys of any female rat killed after 5 or 12 days. These results were in agreement with those of inhalation studies and provide additional evidence that the formation of hyaline droplets in response to exposure to volatile hydrocarbons may be unique to the male rat.

Viscosity effect on the structural compactness of latex flocs formed under weak depletion attractions.

Dilute aqueous dispersions of colloidal polystyrene latex spheres were flocculated by adding a nonadsorbing polymer sample, poly(acrylic acid). The structural compactness of the flocs thus formed was characterized in terms of their mass fractal dimension using the small-angle static light scattering technique. It was found that with low poly(acrylic acid) concentrations and thus weak depletion attraction forces, the dispersion medium viscosity had a marked effect on the floc structure. An increase in the viscosity led to formation of denser flocs. This was revealed in three sets of depletion flocculation experiments: (a) adjusting the background electrolyte concentration at a fixed level of poly(acrylic acid), (b) using water and 30% (w/w) glycerol as the respective solvents, and (c) inducing latex flocculation with two poly(acrylic acids) of different molecular weights at the respective critical polyacid concentrations. Direct force measurements were made with atomic force microscopy to isolate the influence of viscosity on floc structure from that of interparticle interaction energies. We conclude that the formation of denser flocs with increasing medium viscosity can be attributed to the reduced diffusivity of particles in the solution. The latter resulted in an enhanced rate of floc restructuring (through relaxation of attached particles) relative to floc growth.

Septic arthritis of the temporomandibular joint: review of the literature and report of two cases in children.

Septic arthritis of the temporomandibular joint (TMJ) has a high morbidity, is infrequently reported, and has been described almost exclusively in adults. We present two cases of septic arthritis of the TMJ that occurred in children after minor blunt trauma. Literature related to septic arthritis of the TMJ was reviewed, and a composite list of cases was constructed. The most common causes were various infections of the head and neck, rheumatic joint disease, and iatrogenesis. Pathogens may gain access to the TMJ by several routes. Patients typically present with an acute, tender, monarticular arthritis with associated swelling and erythema. Malaise, nausea, and vomiting may also be present. Traumatic effusions, fractures, and neoplasms may present in a similar fashion, and mimic TMJ septic arthritis. Staphylococcus aureus is the most commonly reported pathogen and often causes permanent joint damage. Aspiration and analysis of joint fluid, as well as blood chemistry, imaging studies, and clinical impression, may assist in the diagnosis. Timely diagnosis and treatment are essential for a successful outcome; therapy should include antimicrobial agents, adequate drainage, and resting of the joint. Complications include spread of infection, postinfectious bony changes, and fibrous (or bony) ankylosis of the temporomandibular joint.

Gastric spirillosis in beagles.

Light microscopic, ultrastructural, and microbiologic evaluations were performed on stomachs from 30 healthy laboratory-reared Beagles. Spiral-shaped microorganisms were seen in the gastric glands and parietal cell canaliculi of all the dogs. Organisms were most numerous in the cardia and in the region of the fundic-pyloric junction. Lymphoreticular hyperplasia, dilatation of parietal cell canaliculi, and degeneration of individual parietal cells (rarely seen) were the only morphologic alterations seen. Organisms were helical, had tufts of flagella at each end, and were approximately 0.5 X 7.0 micron; some had a distinct axial fibril (indicating two distinct forms of the organism). Attempts to propagate a viable culture of the organism were not successful. The organism most closely resembled those of the genus Spirillum. Because the organism was commonly found in the gastric mucosa of healthy Beagles, it probably should be considered part of the natural gastric flora of dogs.