Disease associations in alpha-1-antitrypsin deficiency.

INTRODUCTION: In addition to emphysema alpha-1-antitrypsin deficiency (AATD) has been shown to be associated with several inflammatory conditions, including bronchiectasis, vasculitis, (in particular Wegener's granulomatosis), and panniculitis, suggesting neutrophil proteinases also play a role in their pathophysiology. However, it remains unknown whether other inflammatory diseases are also more prevalent in AATD than the general population. The current study describes the prevalence of other co-morbidities in AATD with particular emphasis on inflammatory bowel disease. METHODS AND RESULTS: The case notes of 651 PiZZ or PiZnull patients attending the UK national centre for AATD between 1996 and 2011 were reviewed. The prevalence of inflammatory bowel disease (1.5%) was higher than that predicted in the UK (0.4%). Ten patients had a confirmed diagnosis of ulcerative colitis, and 1 had Crohn's disease. In 2 cases there was a family history of inflammatory bowel disease and all but 1 patient were ex or never smokers. There was also a higher prevalence of hypothyroidism in this patient group than expected for the UK population - 26 cases (7.2% of females and 1.3% of males). CONCLUSIONS: The current study of the UK cohort of patients with AATD confirmed a higher prevalence of ulcerative colitis than would be expected in the general population, providing further evidence of a potential link between these 2 conditions. In addition, the data suggested a potential link between hypothyroidism and AATD.

A review of dental implants and infection.

Dental implants have become increasingly common for the management of tooth loss. Despite their placement in a contaminated surgical field, success rates are relatively high. This article reviews dental implants and highlights factors leading to infection and potential implant failure. A literature search identified studies analysing the microbial composition of peri-implant infections. The microflora of dental peri-implantitis resembles that found in chronic periodontitis, featuring predominantly anaerobic Gram-negative bacilli, in particular Porphyromonas gingivalis and Prevotella intermedia, anaerobic Gram-negative cocci such as Veillonella spp. and spirochaetes including Treponema denticola. The role of Staphylococcus aureus and coagulase-negative staphylococci that are typically encountered in orthopaedic infections is debatable, although they undoubtedly play a role when isolated from clinically infected sites. Likewise, the aetiological involvement of coliforms and Candida spp. requires further longitudinal studies. Currently, there are neither standardised antibiotic prophylactic regimens for dental implant placement nor universally accepted treatment for peri-implantitis. The treatment of infected implants is difficult and usually requires removal. In the UK there is no systematic post-surgical implant surveillance programme. Therefore, the development of such a project would be advisable and provide valuable epidemiological data.

Clinical investigation of the novel iron-chelating agent, CP94, to enhance topical photodynamic therapy of nodular basal cell carcinoma.

BACKGROUND: Photodynamic therapy (PDT) involves the activation of a photosensitizer by visible light to produce activated oxygen species within target cells, resulting in their destruction. Evidence-based guidelines support the efficacy of PDT using topical 5-aminolaevulinic acid (ALA-PDT) in actinic keratoses, Bowen disease and basal cell carcinoma (BCC). Efficacy for nodular BCC appears inferior to that for superficial BCC unless prior debulking or repeat treatments are performed. Objectives The aim of this study was to assess the safety and efficacy of adding a novel iron-chelating agent, CP94 (1,2-diethyl-3-hydroxypyridin-4-one hydrochloride), to topical ALA, to temporarily increase the accumulation of the photosensitizer in the tumour. METHODS: A mixed topical formulation of ALA + increasing concentrations of CP94 was used to carry out PDT on previously biopsied nodular BCC with no prior lesion preparation using standard light delivery. The area was assessed clinically and surgically excised 6 weeks later for histological examination. RESULTS: Enhanced PDT using 40% CP94 resulted in significantly greater clearance rates in nodular BCC than with ALA-PDT alone, in our protocol of single-treatment PDT with no lesion preparation. CONCLUSIONS: The results of this study demonstrate the safe and effective use of an enhanced ALA-PDT protocol for nodular BCC using CP94, with no adverse reactions to this modification. This is the first time this formulation has been used in patients. This formulation is now the focus of further study.

Effect of storage and postage on recovery and quantitation of bacteria in sputum samples.

AIMS: The aims of the study were to compare bacterial recovery following storage of sputum samples at 20 degrees C room temperature and 4 degrees C (refrigerated) for 24 h, and to determine the effect of postage on viable bacterial numbers. METHODS: A total of 38 individual sputum samples from clinically stable patients with bronchiectasis were split into three equal aliquots and quantitative bacterial culture was performed (i) immediately, (ii) following storage at 4 degrees C for 24 h or (iii) following storage at 20 degrees C for 24 h. A further 42 sputum samples were split into two equal aliquots and quantitative bacterial culture was performed either immediately or following postage back to the laboratory by first-class mail from an outside location. RESULTS: The predominant organism could still be recovered following storage at 4 degrees C and 20 degrees C, but viable numbers were significantly reduced following storage at 4 degrees C (p<0.004) by at least an order of magnitude (10-fold) in 24% of samples stored at 4 degrees C compared with only 8% stored at 20 degrees C. Posting samples back to the laboratory did not affect the recovery of bacterial species and there was no difference in viable numbers isolated. CONCLUSIONS: The results suggest that storage at room temperature is preferable to refrigeration as it retains the species isolated and the viable number. The data also confirm that sputum samples can be posted to the laboratory from patients in the community without affecting qualitative or quantitative results.

Relationship of sputum color to nature and outpatient management of acute exacerbations of COPD.

STUDY OBJECTIVES: To stratify COPD patients presenting with an acute exacerbation on the basis of sputum color and to relate this to the isolation and viable numbers of bacteria recovered on culture. DESIGN: Open, longitudinal study of sputum characteristics and acute-phase proteins. SETTING: Patients presenting to primary-care physicians in the United Kingdom. Patients were followed up as outpatients in specialist clinic. PATIENTS: One hundred twenty-one patients with acute exacerbations of COPD were assessed together with a single sputum sample on the day of presentation (89 of whom produced a satisfactory sputum sample for analysis). One hundred nine patients were assessed 2 months later when they had returned to their stable clinical state. INTERVENTIONS: The expectoration of green, purulent sputum was taken as the primary indication for antibiotic therapy, whereas white or clear sputum was not considered representative of a bacterial episode and the need for antibiotic therapy. RESULTS: A positive bacterial culture was obtained from 84% of patients sputum if it was purulent on presentation compared with only 38% if it was mucoid (p < 0.0001). When restudied in the stable clinical state, the incidence of a positive bacterial culture was similar for both groups (38% and 41%, respectively). C-reactive protein concentrations were significantly raised (p < 0.0001) if the sputum was purulent (median, 4.5 mg/L; interquartile range [IQR], 6. 2 to 35.8). In the stable clinical state, sputum color improved significantly in the group who presented with purulent sputum from a median color number of 4.0 (IQR, 4.0 to 5.0) to 3.0 (IQR, 2.0 to 4. 0; p < 0.0001), and this was associated with a fall in median C-reactive protein level to 2.7 mg/L (IQR, 1.0 to 6.6; p < 0.0001). CONCLUSIONS: The presence of green (purulent) sputum was 94.4% sensitive and 77.0% specific for the yield of a high bacterial load and indicates a clear subset of patient episodes identified at presentation that is likely to benefit most from antibiotic therapy. All patients who produced white (mucoid) sputum during the acute exacerbation improved without antibiotic therapy, and sputum characteristics remained the same even when the patients had returned to their stable clinical state.

Sequential nucleic acid and recombinant adenovirus vaccination induces host-protective immune responses against Taenia ovis infection in sheep.

Sheep were immunized with a protective recombinant antigen (45W) from the cestode parasite Taenia ovis using three different vaccine delivery systems, either alone or in different combinations. The DNA encoding 45W was cloned into the expression plasmid pcDNA 3 and an ovine adenovirus to create nucleic acid and recombinant viral vector vaccines, respectively. Sheep received two vaccinations with various combinations of these two delivery systems and/or purified recombinant 45W protein in a conventional vaccine formulation containing Quil A as adjuvant (protein/Quil A vaccine). Sheep receiving two inoculations of either the nucleic acid or the recombinant adenovirus alone, demonstrated only low levels of 45W-specific antibody. However, immunization with either nucleic acid or recombinant adenovirus primed animals to mount an enhanced immune response after a subsequent vaccination with the protein/ Quil A vaccine. The most striking result was that sheep initially immunized with the nucleic acid vaccine and boosted with the recombinant adenovirus, mounted IgG1 responses > 65 fold higher than those of sheep receiving either vaccine alone. The level of antibody in these sheep was commensurate with that observed in animals vaccinated twice with the protein/Quil A adjuvanted vaccine. In both cases, host-protection from experimental challenge infection with T. ovis was obtained.

Comparison of field and vaccine strains of Australian fowlpox viruses.

The mild fowlpox vaccine, FPV M, widely used in Australia is composed of two predominant genotypes based upon differences identifiable in restriction enzyme analyses of plaque purified derivatives of this vaccine. The differences, where identifiable, were in the end fragments of the genomes. Five field isolates of FPV from chickens in New South Wales showed restriction enzyme profiles closely related to the more virulent (standard) vaccine strain, FPV S. The FPV S strain differs from FPV M in both terminal genome fragments and in the presence of a PstI fragment of approximately 10kb (this fragment was also present in PstI digests of all of the field isolates). Plaque purified derivatives of FPV M showed similar lesion development upon inoculation into the wing web of chickens. The field isolates showed significantly higher virulence in day-old and three-week-old chickens in comparison with FPV M. One field isolate was similar to the FPV S vaccine. Two isolates had slowly developing wing web lesions, caused significant secondary lesions in three-week-old chickens and generalised poxvirus infection when inoculated into day-old chickens. For two isolates, the primary wing web lesion took even longer to develop and resolve although these isolates did not cause generalised poxvirus infection. It was possible to identify four virulence/pathogenicity types amongst these vaccine and field isolates of FPV. These strains may allow the characterisation of FPV encoded virulence factors. The field strains with higher virulence may be suitable as parent strains for the construction of FPV recombinants with enhanced immune responses to co-expressed vaccine antigens when compared with current FPV M strain based recombinants.

Vaccinia virus-expressed bovine ephemeral fever virus G but not G(NS) glycoprotein induces neutralizing antibodies and protects against experimental infection.

Two related glycoproteins (G and G(NS)) encoded in the bovine ephemeral fever virus (BEFV) genome were expressed from recombinant vaccinia viruses (rVV). Both proteins were detected in lysates of rVV-infected cells by labelling with D-[6-3H]glucosamine or by immuno-blotting. The recombinant G protein (mol. mass 79 kDa) appeared slightly smaller than the native G protein but reacted with monoclonal antibodies directed against all defined neutralizing antigenic sites (G1, G2, G3a, G3b and G4). The recombinant G(NS) protein (mol. mass 90kDa) was identical in size to the native G(NS) protein and failed to react by immuno-fluorescence with anti-G protein monoclonal or poly-clonal antibodies. Antisera raised in rabbits against rVV-G or rVV-G(NS) both reacted strongly by immuno-fluorescence and immuno-electron microscopy with BEFV-infected cells. The G protein was localized intracellularly in the endoplasmic reticulum/Golgi complex and at the cell surface associated with budding and mature virus particles. The G(NS) protein also localized intracellularly in the endoplasmic reticulum/Golgi complex; however, at the cell surface it was associated with amorphous structures and not with budding or mature virions. Rabbits vaccinated with rVV-G developed high levels of antibodies which neutralized BEFV grown in either mammalian or insect cells. Cattle vaccinated with rVV-G also produced neutralizing antibodies and were protected against experimental BEFV infection. In contrast, rVV-G(NS) vaccinated rabbits and cattle failed to produce neutralizing antibodies and, after challenge, BEFV was isolated from two-thirds of the vaccinated cattle.

Dynamics of respiratory tract colonization by Branhamella catarrhalis in bronchiectasis.

Branhamella catarrhalis is increasingly recognized as a lower respiratory tract pathogen, particularly in chronic lung diseases. This project defines a population of patients in whom the dynamics of colonization and infection caused by this organism could be studied. A method employing pulsed field gel electrophoresis (PFGE) of genomic DNA was developed. Twenty-eight patients with bronchiectasis followed prospectively for 26.8 mo (mean) were seen monthly or bimonthly and at the time of a purulent exacerbation. Quantitative bacterial cultures were performed on sputum obtained at each visit. Six of 28 had B. catarrhalis isolated repeatedly. Viable numbers of B. catarrhalis were similar to other bacterial pathogens. Restriction fragment length polymorphism (RFLP) analysis of chromosomal DNA using PFGE was performed on 37 of the 47 isolates recovered. Each patient was colonized by two to four strains with different RFLP patterns. Duration of colonization by the same strain was 2.3 mo (mean). Strain acquisition did not correlate with exacerbation, antibiotic therapy, or season. We conclude that (1) a subset of bronchiectatic patients is colonized with B. catarrhalis, (2) RFLP is a sensitive tool to study strain acquisition, and (3) acquisition and clearance of B. catarrhalis from the respiratory tract is a dynamic process.

In vivo study of indomethacin in bronchiectasis: effect on neutrophil function and lung secretion.

Bronchiectasis is associated with sputum containing high levels of the proteolytic enzyme elastase, which is thought to be involved in the pathogenesis of the disease. Agents which inhibit neutrophil function and interfere with neutrophil elastase release may have a beneficial effect on the development and progression of such diseases. We have studied the effects of the nonsteroidal anti-inflammatory agent indomethacin on neutrophil function in nine patients with clinically stable bronchiectasis. All patients remained clinically stable during the study. We observed a significant reduction in peripheral neutrophil chemotaxis to 10 nmol.L-1 N-formyl-methionyl-leucyl-phenylalanine (FMLP) from a mean of 19.86 (SEM 1.35) to 8.46 (0.68) cells.field-1 after 4 weeks of therapy. There was also a significant reduction in fibronectin degradation both by resting and FMLP-stimulated neutrophils, from a mean of 1.90 (0.19) micrograms x 3 x 10(5) cells at the start of therapy to 0.87 (0.08) micrograms after 4 weeks, and from 3.17 (0.35) micrograms to 1.48 (0.05) micrograms, respectively. There was no effect on spontaneous or stimulated superoxide anion generation by neutrophils. Despite the marked changes in peripheral neutrophil function, no adverse effect was observed on viable bacterial load in the bronchial secretions. In addition, there was no difference in sputum albumin, elastase or myeloperoxidase levels, and only minor changes in the chemotactic activity of the sputum. These results suggest that nonsteroidal anti-inflammatory agents have a major effect on peripheral neutrophil function but do not appear to have an adverse effect on bacterial colonization of the airways.

Simple method for quantifying viable bacterial numbers in sputum.

AIMS: To establish a simple method of quantitative culture for determining the viable bacterial numbers present in expectorated sputum samples. METHODS: Sputum samples were homogenised with dithiothreitol, sterile saline or glass beads to determine which method recovered the greatest number of viable bacteria. Culture broths were also incubated with dithiothreitol and sampled over time to determine its effect on bacterial viability. Sputum samples homogenised with dithiothreitol were diluted in sterile saline and sampled using either standard bacteriological loops or a precision pipette to determine which method resulted in the least variation. RESULTS: Homogenisation of sputum using dithiothreitol increased the recovery of viable bacteria compared with sterile glass beads and/or saline, with no apparent effect on bacterial viability when incubated with culture broths. By inoculating agar plates with 10(-3), 10(-4) and 10(-5) dilutions of the homogenised sputum sample, all potential pathogens could easily be identified. A 10 microliter sample volume dispensed by precision pipette and spread with a "hockey stick" resulted in the least variation between plates (less than 16%) and an even distribution of bacterial colonies. Numbers of viable bacteria recovered from different aliquots of individual sputum samples were generally of the same order of magnitude. CONCLUSIONS: This method represents a relatively quick and simple technique for accurately quantifying viable bacteria present in sputum samples. The use of a small portion appears to be representative of the sample as a whole.

The isolation and characterization of non-typeable Haemophilus influenzae from the sputum of adult cystic fibrosis patients.

The role of non-typeable Haemophilus influenzae in cystic fibrosis (CF) remains unclear. We wanted, therefore, to determine the presence and characteristics of non-typeable H. influenzae in sputum samples from patients with CF. In order to do this, we have assessed sputum samples from 55 consecutive clinically stable patients seen routinely at an adult CF out-patient clinic. Quantitative bacterial culture was performed using a selective media containing cefsoludin, and isolates were characterized by biotyping and outer membrane protein profile analysis. In 17 (30%) of these samples, non-typeable H. influenzae was isolated and was present in similar viable numbers (mean 7.7 x 10(8) colony-forming units (cfu).mL-1; SEM 3.1) to Pseudomonas aeruginosa (mean 8 x 10(8) cfu.mL-1: SEM 2.4). All non-typeable H. influenzae isolates recovered were beta-lactamase negative and sensitive to a range of antibiotics. Several biotypes and outer membrane protein profiles were observed, with no apparent association between these two phenotypic characteristics. The study showed that large numbers of non-typeable H. influenzae are often present in sputum from adult patients with CF. Further longitudinal studies of outer-membrane protein profile analysis are required to determine the dynamics of non-typeable H. influenzae colonization in individual patients and the clinical significance.

Retrovirus-like particles produced by vaccinia viruses expressing gag-pro-pol region genes of bovine leukaemia virus.

Processing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rVVs) expressing regions of the bovine leukaemia virus genome. Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rVV carrying the gag and truncated protease (pro) gene. Processing of Pr44 was observed after infection of cells with a rVV carrying the gag and pro gene or a rVV expressing the gag, pro and polymerase (pol) gene. Reverse transcriptase activity was detected only in association with particles produced by gag-, pro- and pol-expressing recombinants. Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane. Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rVVs expressing gag and pro or gag, pro and pol formed mature particles.

Characterisation of Australian ovine adenovirus isolates.

We have characterised two groups of adenoviruses isolated from sheep in Australia. Restriction endonuclease maps for enzymes BamHI, ClaI, SalI, SmaI and SphI have been determined for the genome of ovine adenoviruses related to bovine adenovirus serotype 7 (BAV 7) from sheep in Western Australia. Although previously serotyped as BAV 7 these isolates are different from bovine isolates of BAV 7 based on comparison with published restriction endonuclease profiles and maps of BAV 7 cattle isolates. Additional adenovirus isolates obtained from Victorian sheep have been serotyped as ovine adenovirus type 5 (OAV 5). On the basis of restriction endonuclease analysis these viruses are different from the sheep BAV 7 isolates. Following infection of sheep with ovine BAV 7 and OAV 5 isolates, virus was recovered from nasal and rectal swabs for several days. Antibodies detected by ELISA and serum neutralisation tests (SN) developed by 15 days after infection. Virus also spread from the infected sheep to an incontact control and one of ten sheep purchased for infection studies had SN antibodies to BAV 7 suggesting that BAV 7-like viruses naturally infect sheep in Victoria and Western Australia. With further development, these ovine adenoviruses may be suitable as vectors for the delivery of vaccine antigens to sheep and cattle.

Sputum and serum pharmacokinetics of loracarbef (LY163892) in patients with chronic bronchial sepsis.

Sputum and serum pharmacokinetics of loracarbef (LY163892) were performed in 19 patients with purulent bronchiectasis. Nine of the patients received 200 mg twice daily and ten patients, 400 mg twice daily, for a total of 14 days. beta-Lactamase activity was measurable in the lung secretions of all 19 patients at the start of therapy. Mean peak serum concentrations of 11.7 mg/L (S.E.M. 1.7) were recorded at 1 h after administration of 200 mg doses on day 2 of therapy and were 18.5 mg/L (S.E.M. 1.9) at 1.5 h in the 400 mg group. Loracarbef was shown to penetrate lung secretions even in the presence of beta-lactamase activity. Mean peak sputum concentrations were achieved between 2 and 4 h following dosing and were 0.2 mg/L (S.E.M. 0.05) in the 200 mg group and 0.4 mg/L (S.E.M. 0.08) in the 400 mg group. On days 7 and 14 of therapy, sputum loracarbef concentrations were similar 4 h after the morning dose (0.23 mg/L following 200 mg; 0.35 mg/L after 400 mg). These concentrations were approximately 2% of the peak serum concentration and penetration into lung secretions is similar to other beta-lactams.

Cochlear damage in guinea pigs following contralateral sound stimulation with and without gentamicin.

The effect of a minimally damaging sound exposure and a sub-ototoxic dose of gentamicin on cochlear hair cells contralateral to the sound exposure was evaluated. The cochleae of pigmented guinea pigs exposed to an 8 kHz pure tone at 116 dB SPL for 1 h and/or 50 mg/kg/day of gentamicin for 10 consecutive days and repeated after an interval of 3 weeks, were used for this purpose. Hair cell loss was found to have occurred in the contralateral cochleae following the sound exposure alone. The occurrence of potentiation, synergism and differential synergism between the agents in the contralateral ears was also seen. Possible explanations for these phenomena are proposed.

Interaction between sound and gentamicin: immediate threshold and stereociliary changes.

Our aim was to determine whether the immediate effects of a just-damaging sound exposure (8 kHz at 116 dB SPL for 1 h) might be potentiated by a single sub-ototoxic dose of gentamicin (50 mg kg-1). Auditory brainstem responses in pigmented guinea pigs were measured before and after treatment and used to calculate threshold shift (TS). Histological disturbances to sensory hair cells were assessed by scanning electron microscopy. All experimental ears excepting the gentamicin only group showed TS and histological changes. The largest TSs occurred at half an octave above the exposure frequency, and were greatest in the gentamicin + sound (G + S) group. First row outer hair cells showed most histological disturbances, followed by inner hair cells. The severest histological damage occurred at the exposure frequency and basally from it, the G + S group being most affected. Generally, there was good correlation between the severity of TS and histological damage. Results from both analyses indicated greater changes with gentamicin present.

A comparison of initial and permanent surface changes to guinea pig hair cells after acoustic overstimulation.

Groups of pigmented guinea pigs were exposed unilaterally to a 15 kHz pure tone at 133 dB SPL for 7.5 min. The inner ears of one group were examined by light microscopy to count damaged hair cells 3 weeks after exposure. Four other groups were investigated using scanning electron microscopy to assess the progression of initial surface changes after sound exposure. Hair cells were examined at 0, 10 and 30 min and compared with those seen at 3 weeks. Drastic stereociliary disturbances were present immediately after this short sound stimulation. Changes initially affected the first row of outer hair cells, followed by the inner hair cell row, and then spread to all rows at the centre of a damaged area. These changes appeared more advanced in specimens from the later groups. At 3 weeks, primarily phalangeal scars were seen at the main damaged area, with partially degenerating cells at the periphery. The latter still showed stereociliary disturbances, but the types predominating were different from those seen initially. However, no differences were found in the extens of damage when the various exposure groups were compared. No changes due to overstimulation were present in the contralateral ears.