The association between gut microbiome and growth in infants with cystic fibrosis.

BACKGROUND: In cystic fibrosis (CF), pathophysiologic changes in the gastrointestinal tract lead to malnutrition and altered gut microbiome. Microbiome alterations have been linked to linear growth, gut inflammation and respiratory manifestations. Elucidating these gut microbiome alterations may provide insight into future nutritional management in CF. METHODS: Infants were followed for 12-months at four sites in the United States (US-CF) and Australia (AUS-CF). 16S rRNA gene sequencing was performed on longitudinal stool samples. Associations between microbial abundance and age, antibiotic prophylaxis, malnutrition, and breast feeding were evaluated using generalized linear mixed models. Taxonomic and predictive functional features were compared between groups. RESULTS: Infants with CF (N = 78) were enrolled as part of a larger study. AUS-CF infants had higher mean weight-for-age z-scores than US-CF infants (p = 0.02). A subset of participants (CF N = 40, non-CF disease controls N = 10) provided stool samples for microbiome analysis. AUS-CF infants had lower stool alpha diversity compared to US-CF infants (p < 0.001). AUS-CF infants had higher relative abundance of stool Proteobacteria compared to US-CF infants which was associated with antibiotic prophylaxis (p < 0.001). Malnutrition (weight-for-age <10th percentile) was associated with depleted Lactococcus (p < 0.001). Antibiotic prophylaxis (p = 0.002) and malnutrition (p = 0.012) were linked with predicted decreased activity of metabolic pathways responsible for short chain fatty acid processing. CONCLUSIONS: In infants with CF, gut microbiome composition and diversity differed between the two continents. Gut microbial diversity was not linked to growth. The relationship between malnutrition and antibiotic prophylaxis with reduced SCFA fermentation could have implications for gut health and function and warrants additional investigation.

Experimental and Monte Carlo determination of the TG-43 dosimetric parameters for the model 9011 THINSeed brachytherapy source.

PURPOSE: AAPM TG-43 brachytherapy dosimetry parameters for a new, smaller diameter 1251 brachytherapy source (THINSeed, model 9011) were determined using LiF:Mg,Ti thermoluminescent dosimeter (TLD-100) microcubes and Monte Carlo simulations. METHODS: Two polymethyl methacrylate phantoms were machined to hold TLD-100 microcubes at specific locations for the experimental determination of the radial dose function, dose-rate constant, and anisotropy functions of the new source. The TG-43 parameters were also calculated using Monte Carlo simulations. For comparison, the model 6711 source was also investigated. RESULTS: Experimental results for both models 9011 and 6711 sources showed good agreement with Monte Carlo values, as well as with previously published values. CONCLUSIONS: The TG-43 parameters for the new source model are similar to those of model 6711; however, they represent two separate sources and TG-43 parameters used in treatment planning must be source specific.

Teratogenicity of vitamin B6 deficiency: omphalocele, skeletal and neural defects, and splenic hypoplasia.

Vitamin B(6) deficiency was induced in pregnant rats with a deficient diet and with 4-deoxypyridoxine, a B(6) antagonist. Treated animals developed typical skin changes of B(6) deficiency. Fetuses were small and appeared anemic. Major fetal malformations were omphalocele, exencephaly, cleft palate, micrognathia, digital defects, and splenic hypoplasia. This teratologic system was developed as a model for human syndromes that exhibit combined immunologic and neurologic or skeletal defects.

Observational evidence for an active surface reservoir of solid carbon dioxide on Mars.

High-resolution images of the south polar residual cap of Mars acquired in 1999 and 2001 show changes in the configuration of pits, intervening ridges, and isolated mounds. Escarpments have retreated 1 to 3 meters in 1 martian year, changes that are an order of magnitude larger than can be explained by the sublimation of water ice, but close to what is expected for sublimation of carbon dioxide ice. These observations support a 35-year-old conjecture that Mars has a large surface reservoir of solid carbon dioxide. The erosion implies that this reservoir is not in equilibrium with the present environment and that global climate change is occurring on Mars.

Extracellular purines are biomarkers of neutrophilic airway inflammation.

Purinergic signalling regulates airway defence mechanisms, suggesting that extracellular purines could serve as airway inflammation biomarkers in cystic fibrosis (CF). The purines adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine were measured in sputum from 21 adults (spontaneously expectorated from seven CF patients, induced from 14 healthy controls) to assess normal values and CF-associated changes. Subsequently, purine levels were measured in bronchoalveolar lavage fluid (BALF) from 37 children (25 CF patients, 12 disease controls) and compared with neutrophil counts, presence of airway infection and lung function. To noninvasively assess airway purines, ATP levels were measured using luminometry in exhaled breath condensate (EBC) from 14 children with CF and 14 healthy controls, then 14 CF children during a pulmonary exacerbation. Both ATP and AMP were elevated in sputum and BALF from CF subjects compared with controls. In BALF, ATP and AMP levels were inversely related to lung function and strongly correlated with neutrophil counts. In EBC, ATP levels were increased in CF relative to controls and decreased after treatment of CF pulmonary exacerbation. The purines adenosine triphosphate and adenosine monophosphate are candidate biomarkers of neutrophilic airways inflammation. Measurement of purines in sputum or exhaled breath condensate may provide a relatively simple and noninvasive method to track this inflammation.

LiF:Mg,Ti TLD response as a function of photon energy for moderately filtered x-ray spectra in the range of 20-250 kVp relative to 60Co.

The response of LiF:Mg,Ti thermoluminescent dosimeters (TLDs) as a function of photon energy was determined using irradiations with moderately filtered x-ray beams in the energy range of 20-250 kVp relative to the response to irradiations with 60Co photons. To determine if the relative light output from LiF:Mg,Ti TLDs per unit air kerma as a function of photon energy can be predicted using calculations such as Monte Carlo (MC) simulations, measurements from the x-ray beam irradiations were compared with MC calculated results, similar to the methodology used by Davis et al. [Radiat. Prot. Dosim. 106, 33-43 (2003)]. TLDs were irradiated in photon beams with well-known air kerma rates using the National Institute of Standards and Technology traceable M-series x-ray beams in the range of 20-250 kVp. For each x-ray beam, several sets of TLDs were irradiated for times corresponding to different air kerma levels to take into account any dose nonlinearity. TLD light output was then compared to that from several sets of TLDs irradiated at similar corresponding air kerma levels using a 60Co irradiator. The MC code MCNP5 was used to account for photon scatter and attenuation in the holder and TLDs and was used to calculate the predicted relative TLD light output per unit air kerma for irradiations with each of the experimentally used photon beams. The measured relative TLD response as a function of photon energy differed by up to 13% from the MC calculations. We conclude that MC calculations do not accurately predict the relative response of TLDs as a function of photon energy, consistent with the conclusions of Davis et al. [Radiat. Prot. Dosim. 106, 33-43 (2003)]. This is likely due to complications in the solid state physics of the thermoluminescence process that are not incorporated into the simulation.

Immunologic responses to bacteriophage phi-X 174 in immunodeficiency diseases.

Immunologic responses to bacteriophage varphiX 174 were studied in 26 patients with immunodeficiency diseases. In eight cases of infantile X-linked agammaglobulinemia, there was prolonged circulation of phage and no detectable antibody response. The remaining 18 patients cleared phage normally and produced antibodies. 10 of these patients made only IgM antibody in spite of repeated immunization; all of these have recurrent respiratory tract infections and require treatment with gamma globulin and antibiotics. Eight patients made both IgM and IgG antibody; they experience either milder or no infections, and only one requires treatment with gamma globulin. Prolonged circulation of bacteriophage varphiX 174 and the absence of a detectable antibody response appear to be distinguishing characteristics of X-linked agammaglobulinemia if severe combined immunodeficiency can be excluded.

Experimental bacterial keratitis: a quantitative model of leukocyte migration following transfusion.

A model for the study of polymorphonuclear leukocyte (PMN) migration after transfusion employing induction of keratitis in guinea pigs was developed. Initial studies demonstrated that compared with other agents, intracorneal injection of Pseudomonas aeruginosa following in vivo labelling of PMN by administration of 3H-thymidine produced the greatest influx of radiolabelled PMN into corneas. In subsequent studies, donor peritoneal PMN were radio-labelled by injection of donors with 3H-thymidine. Neutropenia was induced in recipients by whole body irradiation, and they were infected intracorneally with Pseudomonas prior to transfusion. Corneal radioactivity was assayed 24 h after induction of keratitis and the number of donor PMN in corneas was calculated. Half-life of transfused PMN in non-neutropenic recipients was 1.9 h. Arrival of labelled PMN at infected corneas in recipient animals ranged between 0.1-1.0% of transfused cells. Exposure of donor PMN to sonication or to 45 degrees C for 20 min reduced the proportion of PMN arriving at infected corneas (P less than 0.001). Storage of PMN for 24 h at 4 degrees C led to a greater ingress of donor PMN compared with storage at 37 degrees C (P less than 0.01). This model allows quantitation of in vivo PMN function after transfusion and should allow assessment of the effects of most aspects of PMN transfusion technique upon such function.

Control of pathogenic effector T-cell activities in situ by PD-L1 expression on respiratory inflammatory dendritic cells during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is a leading cause of severe lower respiratory tract illness in young infants, the elderly and immunocompromised individuals. We demonstrate here that the co-inhibitory molecule programmed cell death 1 (PD-1) is selectively upregulated on T cells within the respiratory tract during both murine and human RSV infection. Importantly, the interaction of PD-1 with its ligand PD-L1 is vital to restrict the pro-inflammatory activities of lung effector T cells in situ, thereby inhibiting the development of excessive pulmonary inflammation and injury during RSV infection. We further identify that PD-L1 expression on lung inflammatory dendritic cells is critical to suppress inflammatory T-cell activities, and an interferon-STAT1-IRF1 axis is responsible for increased PD-L1 expression on lung inflammatory dendritic cells. Our findings suggest a potentially critical role of PD-L1 and PD-1 interactions in the lung for controlling host inflammatory responses and disease progression in clinical RSV infection.

Bacteriologic cure of experimental Pseudomonas keratitis.

Two long-term therapy trials with high concentrations of antibiotic were carried out to determine the duration of therapy required to achieve bacteriologic cure of experimental Pseudomonas keratitis in guinea pigs. In the first study, corneas still contained Pseudomonas after 4 days of continual topical therapy with either tobramycin 400 mg/ml, amikacin 250 mg/ml, ticarcillin 400 mg/ml, or carbenicillin 400 mg/ml. In an 11-day trial of topical therapy with tobramycin 20 mg/ml, 34 of 36 corneas grew no Pseudomonas after 6 or more days of therapy. The bacteriologic response to therapy in this model occurred in two phases. About 99.9% or more of the organisms in the cornea were killed in the first 24 hr of therapy. The numbers of bacteria remaining in the cornea declined gradually over the next several days until the corneas were sterile. Optimal antibiotic therapy may include two stages: initial intensive therapy with high concentrations of antibiotic applied frequently to achieve a large rapid decrease in numbers of organisms in the cornea, followed by prolonged, less intensive therapy to eradicate organisms and prevent relapse.

Experimental Pseudomonas keratitis in the rabbit: bacteriologic, clinical, and microscopic observations.

Uniformly severe corneal infections were produced in rabbits by intracorneal injection of a few viable Pseudomonas aeruginosa. The bacteria multiplied rapidly, and within 24 hr, about 10 million organisms were present. The numbers remained stable thereafter. Polymorphonuclear leukocytes (PMNs) began to infiltrate peripheral stroma 24 hr after inoculation. By 32 hr, ring-shaped dense accumulations of PMNs were apparent in the anterior stroma with moderate stromal edema. By 48 hr, the anterior one third of central stroma was severely involved with abscess formation and loss of epithelium, and PMNs had invaded full corneal thickness. The area of liquefactive necrosis eventually involved the entire cornea from limbus to limbus, and collagen staining was lost. Transmission electron microscopy revealed the accumulation of small electron-dense particles in association with collagen fibrils and degranulating PMNs.

Pathogenesis of experimental Pseudomonas keratitis in the guinea pig: bacteriologic, clinical, and microscopic observations.

Uniformly severe corneal infections were produced in guinea pigs by intracorneal injection of about 10 viable Pseudomonas aeruginosa. After a brief lag period, multiplication of bacteria was rapid, reaching geometric means of 280,000 after 24 hr and of 5 million after 48 hr. Within 8 hr after inoculation, polymorphonuclear leukocytes (PMNs) began to infiltrate the anterior two thirds of the stroma. Stromal cells adjacent to the injection site became necrotic and appeared to be engulfed by PMNs. By 14 to 16 hr, an abscess containing a dense aggregate of PMNs and multiplying bacteria developed in the central stroma. By 16 to 24 hr, collagen breakdown was apparent within and around the abscess. Ultrastructural evidence of collagen breakdown included loss of intact collagen fibrils, tactoid formation, and accumulation of amorphous electron-dense material. The area of liquefactive necrosis gradually enlarged, and many corneas perforated after 3 to 4 days. Because the course of infection is highly reproducible, this model should prove useful for many studies of experimental Pseudomonas keratitis.

Septic pulmonary embolism complicating a central venous catheter.

Bacteremia is a recognized complication in patients with indwelling central venous catheters. More recently pulmonary embolism in such patients has also been described. Despite abundant clinical experience with these devices, to our knowledge, septic pulmonary embolism has not been reported in adult patients. This case illustrates such a complication.

Polymorphonuclear leukocyte kinetics in experimentally induced keratitis.

The movement of polymorphonuclear leukocytes (PMNLs) into inflamed corneas was studied using a quantitative technique to measure PMNL chemotaxis in vivo. Our studies suggested that, in this model, most PMNLs enter the cornea through limbal vessels. A variety of bacterial agents, including viable bacteria, killed bacteria, culture filtrates, and endotoxin, were found to induce a significant corneal inflammatory response. Of the agents tested, viable Pseudomonas aeruginosa produced greatest inflammation. Host factors (serum, PMNLs) also induced movement of PMNLs into corneas, but only after preincubation with activating agents. Normal serum, resting PMNLs, and PMNL lysates derived from resting cells did not promote PMNL corneal ingress. These studies provide further insight into the movement of PMNLs into the inflamed cornea and information that may be of use in developing techniques to inhibit the corneal inflammatory response.

Topical tobramycin therapy of experimental Pseudomonas keratitis: an evaluation of some factors that potentially enhance efficacy.

We evaluated several therapeutic modifications in an attempt to improve efficacy of topical therapy with tobramycin of experimental Pseudomonas keratitis in guinea pigs. Removal of corneal epithelium enhanced efficacy of topical therapy with 0.3 mg/ml and 3 mg/ml tobramycin sulfate but did not influence therapy with 40 mg/ml or 400 mg/ml tobramycin. The highest concentration of antibiotic was the most effective; 7 of 12 infected corneas treated with 400 mg/ml tobramycin were sterile in 48 hours. Therapy begun soon after the infection was established, when there were relatively few organisms present, was more effective than therapy begun later, when there were many more bacteria in the cornea. Our results are consistent with a basic therapeutic concept. The most effective regimen is one that achieves the highest safe concentration of antibiotic at the site of infection as early in the course of infection as possible.

Corticosteroid in experimentally induced Pseudomonas keratitis: failure of prednisolone to impair the efficacy of tobramycin and carbenicillin therapy.

The effect of prednisolone on tobramycin and carbenicillin therapy for experimentally induced Pseudomonas keratitis was evaluated. Results were assessed quantitatively by determining the number of bacteria that survived in the cornea. Simultaneous administration of prednisolone did not adversely alter results of treatment with carbenicillin or tobramycin. In another trial, pretreatment with prednisolone for 48 hours before antibiotic therapy was begun did not change significantly the results of therapy with intramuscular tobramycin or carbenicillin. We conclude that corticosteroid therapy does not affect adversely results of antibiotic therapy with tobramycin or carbenicillin in this experimental model.

Staphylococcal keratitis. Experimental model in guinea pigs.

An experimental model of staphylococcal keratitis in guinea pigs was devised that is suitable for quantitative evaluation of therapy. The growth curve in the cornea of a virulent strain of Staphylococcus aureus was determined. The organism multiplied rapidly, reached a peak in about 12 hours, and began to decline in numbers after three days. Infections were relatively resistant to therapy begun 24 hours after infection was established. Treatment started earlier when fewer bacteria were present was more effective than treatment begun later. Treatment begun at the time of infection, which might be considered prophylaxis, was highly effective. When treatment was begun eight hours after infection, tobramycin sulfate and gentamicin sulfate solutions administered topically in doses of 20 mg/ml were more effective than topical bacitracin, erythromycin, clindamycin phosphate, or a solution containing polymyxin B sulfate, neomycin sulfate, and gramicidin. Bacitracin and erythromycin ointments were ineffective.

Antibiotic therapy of experimental Pseudomonas keratitis in guinea pigs.

Antibiotic therapy of experimental Pseudomonas keratitis was evaluated quantitatively by determining numbers of viable bacteria in the cornea of guinea pigs. Topically applied carbenicillin disodium, gentamicin sulfate, and tobramycin sulfate were often significantly more effective than topically applied polymyxin B sulfate. Intramuscular therapy with tobramycin was as effective as topical therapy, and the results exhibited less variability. Topical tobramycin every 30 minutes was significantly more effective than topical therapy every 60 minutes. No combination of antibiotics was significantly better than a single effective drug. The concentration of tobramycin in the aqueous correlated more closely to therapeutic efficacy than did the concentration in the cornea. Although all antibiotics reduced numbers of bacteria in the cornea by more than 99% in the first 24 hours of therapy, none was able to sterilize the cornea in four additional days of continuous therapy. Persistence of organisms despite apparently adequate topical therapy may explain some reported cases of relapse in humans.

Cryotherapy for experimental Pseudomonas keratitis.

The effectiveness of cryotherapy alone and in combination with topical tobramycin sulfate therapy for experimental Pseudomonas keratitis was determined in guinea pigs and rabbits. Results were evaluated quantitatively by determining numbers of viable bacteria surviving in corneas. A brass probe cooled to--79 degrees C and applied directly to infected corneas for six seconds resulted in an immediate 99.9% reduction in bacteria. One freeze-thaw cycle followed by topical tobramycin therapy was significantly more effective than tobramycin therapy alone in five of six strains tested. None of the corneas treated with tobramycin alone demonstrated no growth, whereas 24 of 42 of these infected corneas showed no growth after the combination treatment. We conclude that cryotherapy alone had a rapid bactericdal effect on experimental Pseudomonas keratitis and that it significantly potentiated topical antibiotic therapy for most strains.

Increased susceptibility to infection in experimental xerophthalmia.

Vitamin A-deficient rabbits were used to evaluate the role of secondary bacterial infection in the development of keratomalacia and to describe the resultant clinical and morphologic alterations. The conjunctival sacs of vitamin A-deficient rabbits at different stages of corneal involvement were inoculated with Pseudomonas aeruginosa topically. Approximately two weeks after inoculation, corneal ulceration with stromal melting developed in one of three eyes with severe punctate keratitis and in four of seven eyes with xerosis. Ulceration did not develop in any of the eight eyes with early epithelial graying or mild punctate keratitis. Inflammatory cells (primarily polymorphonuclear leukocytes) infiltrated the anterior corneal stroma of infected corneas. Liquefaction of collagen was observed in association with bacteria alone, as well as in association with polymorphonuclear leukocytes. No signs of infection were observed after conjunctival inoculation of Pseudomonas in the eyes of nine control rabbits.