Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus

Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon-host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild-type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fuily compiemented the ciumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen-coated PMMA. in addition, the cloned clfA gene on a shuttle plasmid aiiowed the weakiy ciumping strain 8325-4 to form clumps with the same avidity as the wild-type strain Newman and also significantly enhanced the adherence of 8325-4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid-phase fibrinogen. The clfA gene encodes a fibrinogen-binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the ceil surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall-associated proteins. Significant homology was found between the ClfA protein and the fibronectin-binding proteins of S. S. aureus, particularly in the N-and C-termini.     

A quantitative microwell assay for chondrocyte cell adhesion

An assay for measuring the quantity of live articular chondrocytes attached to a substratum in microwell plates was established by measuring the absorbance of the blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-dypenyl tetrazolium bromide (MTT). Blue formazan production was optimal at an MTT concentration of 1 mg/ml (100 microliters per microwell) and an incubation period of 3 h. The absorbance of the dye was linearly related to the quantity of cells added per microwell. The number of live chondrocytes attached to adhesive proteins can be quantitated using this technique.     

An immune response defect due to low levels of class II cell surface expression. Analysis of antigen presentation and positive selection.

The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.     

Structure, regulatory polymorphisms, and allelic hypervariability regions in murine I-A alpha

Class II major histocompatibility complex (MHC) molecules, the Ia antigens, are intimately involved in regulating the intensity and specificity of the cellular and humoral responses to T cell-dependent antigens. One approach to understanding the mechanism of this regulation is to analyze the structure and allelic polymorphism of Ia molecules. In addition there are regulatory polymorphisms in the expression of the I-E alpha and I-E beta class II MHC polypeptide chains. Analysis of the cDNA sequence indicates that I-A and I-E alpha chains are similar with short stretches of homology and other regions of nonhomology. Analysis of Northern blots of mRNA indicates that at least three separate types of regulatory polymorphisms result in failure of expression of I-E alpha. Comparison of allelic sequences of six alleles of the I-A alpha chain shows that almost all of the allelic polymorphism is in the first domain and that within the first domain it is clustered in three allelic hypervariable regions within the first domain of I-A alpha. The structural and functional implications of these findings are discussed.     

Disintegration of Dung Pats from Cattle Treated with the Ivermectin Anthelmintic Bolus,or the Biocontrol Agent Duddingtonia flagrans

An experiment was performed during the grazing seasons of 1998, 1999 and 2000 to study the influence of the antiparasitic drug ivermectin and the nematophagous fungus Duddingtonia flagrans on cattle dung disintegration. The faeces originated from groups of animals that were part of a separate grazing experiment where different control strategies for nematode parasite infections were investigated. Each group consisted of 10 first-season grazing cattle that were either untreated, treated with the ivermectin sustained-release bolus, or fed chlamydospores of D. flagrans. Faeces were collected monthly on 4 occasions and out of pooled faeces from each group, 4 artificial 1 kg dung pats were prepared and deposited on nylon mesh on an enclosed pasture and protected from birds. The position of the new set of pats was repeated throughout the 3 years of the study. Each year, the dung pats were weighed 4, 6, 8 and 10 weeks after deposition and immediately afterwards replaced to their initial positions.

Results showed that there was no difference in faecal pat disintegration between groups. However, the time-lag between deposition and complete disintegration of the faeces varied significantly between deposition occasions. Dung pats disappeared within 2 weeks (visual observation) when subjected to heavy rainfall early after deposition, whereas an extended dry period coincided with faeces still remaining 12 months after deposition.

     

Role of Absolute Humidity in the Inactivation of Influenza Viruses on Stainless Steel Surfaces at Elevated Temperatures

Influenza virus has been found to persist in the environment for hours to days, allowing for secondary transmission of influenza via inanimate objects known as fomites. We evaluated the efficacy of heat and moisture for the decontamination of surfaces for the purpose of preventing of the spread of influenza. Aqueous suspensions of influenza A virus were deposited onto stainless steel coupons, allowed to dry under ambient conditions, and exposed to temperatures of 55°C, 60°C, or 65°C and relative humidity (RH) of 25%, 50%, or 75% for up to 1 h. Quantitative virus assays were performed on the solution used to wash the viruses from these coupons, and results were compared with the solution used to wash coupons treated similarly but left under ambient conditions. Inactivation of influenza virus on surfaces increased with increasing temperature, RH, and exposure time. Reductions of greater than 5 logs of influenza virus on surfaces were achieved at temperatures of 60 and 65°C, exposure times of 30 and 60 min, and RH of 50 and 75%. Our data also suggest that absolute humidity is a better predictor of surface inactivation than RH and allows the prediction of survival using two parameters rather than three. Modest amounts of heat and adequate moisture can provide effective disinfection of surfaces while not harming surfaces, electrical systems, or mechanical components, leaving no harmful residues behind after treatment and requiring a relatively short amount of time.In a recent publication, Shaman and Kohn concluded that absolute humidity, which can be calculated if temperature and relative humidity (RH) are known, is the controlling factor in both the inactivation of influenza virus and the transmission of influenza (27). To arrive at this conclusion, Shaman and Kohn reanalyzed experimental data collected by Lowen et al. and Harper (12, 17, 18), which covered a rather narrow range of temperatures typical of normal weather conditions, that is, in the range of 5 to 30°C. For this temperature range, the maximum absolute humidity that can occur is 24 g/m³. One question that comes to mind is whether this trend of decreasing influenza virus survival with increasing absolute humidity (AH) persists as temperature increases. If it does, then AH may also be the controlling factor when heat and moisture are used to decontaminate surfaces. In the present work, we tried to answer this question through a series of experiments in which absolute humidity was sufficiently high to result in effective surface decontamination.Effective and easily implemented public health interventions are needed to prevent the spread of infectious diseases such as influenza. Transmission of influenza virus, especially in the event of a pandemic with a highly virulent strain of influenza virus, such as avian influenza H5N1 virus or 2009 H1N1 influenza A virus, is of great concern due to widespread mortality and morbidity (7, 23). The significant morbidity and mortality associated with seasonal influenza should also not be discounted. There is compelling evidence for transmission of influenza viruses from infected individuals to uninfected individuals by direct contact, via fomites (inanimate objects capable of carrying infectious organisms), and through large droplets expelled during forceful exhalation, such as during coughing and sneezing (2-4, 19). Virtually any exposed surface can become contaminated with infectious viruses and can be a potential source of secondary virus transmission. The probability for transmission increases in situations where many people are in close proximity and in locations with highly transient populations, such as public transportation, air transportation, classrooms, theaters, and other public venues (16, 20, 22). Influenza virus has been found to persist in the environment for hours to days; it has been found on surfaces in day-care centers, hands, laboratory gowns, and in surface dust (3, 4, 9, 29). Efforts to prevent the spread of flu through contact transmission via fomites require methods of decontamination that are easy to use and will not disrupt critical services and operations.Laboratory research has shown that the moisture content of the air is an important factor for antimicrobial survival. Past research on influenza virus has focused on airborne viruses and generally suggests that survival and/or transmission is facilitated by low RH (10, 12, 15, 17, 26, 30). A limited number of surface inactivation studies have been performed at environmentally relevant temperatures, ranging from 7 to 32°C (30), and have shown modest reductions in numbers of influenza viruses (5, 21).We evaluated the efficacy of heat and moisture for decontaminating surfaces and controlling the spread of influenza virus infection. In this study, temperatures were maintained well above room temperature (55 to 65°C) but were still not expected to cause harm to most surfaces, mechanical components, or electrical systems. Furthermore, heat does not leave behind potentially harmful residues, as do most chemical decontaminants, and the use of heat requires a relatively brief period of time for surface treatment and allows resources to be quickly returned to service. To our knowledge no previous measurements of influenza virus inactivation rates on surfaces at these temperatures have been made.     

Thermoregulatory responses to manipulations of photoperiod in wood mice Apodemus sylvaticus from high latitudes (57°N)

1. 1. The thermoregulatory responses to manipulations of photoperiod in wood mice (Apodemus sylvaticus), which were drawn from a population living at a high latitude (57°N) were studied.

2. 2. Mice captured in spring were acclimated to two different photoperiod regimes 16L:8D and 8L:16D at a constant ambient temperature of 24°C, for 3 weeks.

3. 3. Daily rhythms of body temperature, oxygen consumption and body temperature at various ambient temperatures, nonshivering thermogenesis (the response to a noradrenaline injection) and body mass were measured. Minimal overall thermal conductance was calculated for both groups.

4. 4. Acclimation to long photophase increased the thermoregulatory abilities at relatively high ambient temperatures while that of long-scotophase increased thermoregulatory abilities at low ambient temperatures.

5. 5. Changes in photoperiod may therefore be used as cues for seasonal acclimatization of thermoregulatory mechanisms in this population of wood mice.