COVID-19: Extensive epithelial damage and ciliary dyskinesia in hospitalised patients.

Infection with SARS-CoV-2 can cause severe respiratory disease and it is predicted that the COVID-19 pandemic will leave a substantial number of patients with long-term respiratory complications (1).

A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies.

Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system (CNS) function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour.

Test-Retest and Interreader Reproducibility of Semiautomated Atlas-Based Analysis of Diffusion Tensor Imaging Data in Acute Cervical Spine Trauma in Adult Patients.

BACKGROUND AND PURPOSE: DTI is a tool for microstructural spinal cord injury evaluation. This study evaluated the reproducibility of a semiautomated segmentation algorithm of spinal cord DTI. MATERIALS AND METHODS: Forty-two consecutive patients undergoing acute trauma cervical spine MR imaging underwent 2 axial DTI scans in addition to their clinical scan. The datasets were put through a semiautomated probabilistic segmentation algorithm that selected white matter, gray matter, and 24 individual white matter tracts. Regional and white matter tract volume, fractional anisotropy, and mean diffusivity values were calculated. Two readers performed the nonautomated steps to evaluate interreader reproducibility. The coefficient of variation and intraclass correlation coefficient were used to assess test-retest and interreader reproducibility. RESULTS: Of 42 patients, 30 had useable data. Test-retest reproducibility of fractional anisotropy was high for white matter as a whole (coefficient of variation, 3.8%; intraclass correlation coefficient, 0.93). Test-retest coefficient-of-variation ranged from 8.0%-18.2% and intraclass correlation coefficients from 0.47-0.80 across individual white matter tracts. Mean diffusivity metrics also had high test-retest reproducibility (white matter: coefficient-of-variation, 5.6%; intraclass correlation coefficient, 0.86) with coefficients of variation from 11.6%-18.3% and intraclass correlation coefficients from 0.57-0.74 across individual tracts, with better agreement for larger tracts. The coefficients of variation of fractional anisotropy and mean diffusivity both had significant negative relationships with white matter volume (26%-27% decrease for each doubling of white matter volume, P < .01). CONCLUSIONS: DTI spinal cord segmentation is reproducible in the setting of acute spine trauma, specifically for larger white matter tracts and total white or gray matter.

Inner dynein arm defects causing primary ciliary dyskinesia: repeat testing required.

Primary ciliary dyskinesia (PCD) results in chronic nasal symptoms and chest disease leading to bronchiectasis. We noted a number of patients referred for diagnostic testing whose initial results suggested PCD due to an inner dynein arm or radial spoke defect but in whom no abnormality was found on retesting. The present study was an audit of all patients referred for PCD diagnostic testing over a 3-yr period whose initial electron microscopy (EM) and beat pattern analysis suggested an inner dynein arm or radial spoke defect. 21 patients referred for diagnostic testing for PCD suspected of an inner dynein arm defect and six suspected of a radial spoke defect on initial EM and beat pattern analysis had repeat testing performed. On repeat testing, five patients initially suspected of an inner dynein arm defect and one with a radial spoke defect had normal EM and beat pattern, leading to the initial diagnosis being questioned. Patients suspected of PCD due to an inner dynein arm defect or radial spoke defect should have the diagnosis reassessed if it has been based on only one diagnostic sample.

Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia.

Ciliary function studies for the diagnosis of primary ciliary dyskinesia (PCD) are usually performed on nasal brush biopsy samples. It is not uncommon to find disrupted epithelial strips of tissue in these samples, and occasionally throughout a sample. The aim of the present study was to determine if cilia on disrupted ciliated epithelial edges beat with a normal pattern and frequency similar to that of cilia on undisrupted edges. Nasal brush biopsy samples from 42 children in whom the diagnosis of PCD was excluded were assessed. The epithelial strips were categorised into five groups: intact undisrupted ciliated epithelial edge, ciliated epithelial edge with minor projections, ciliated epithelial edge with major projections, an isolated ciliated cell on an epithelial edge and single unattached ciliated cells. Ciliary beat frequency and beat pattern of 50 samples from each group were determined using high speed digital video microscopy. The cilia on epithelial edges with varying degrees of disruption showed significantly reduced beat frequency and significantly increased dyskinesia compared with those on intact, undisrupted ciliated epithelial edges. Ideally, the assessment of ciliary beat pattern and frequency for PCD diagnosis should only be performed on undisrupted ciliated edges.

Recovery of the ciliated epithelium following acute bronchiolitis in infancy.

BACKGROUND: Little is known about the longitudinal changes in the ciliated respiratory epithelium of infants following viral bronchiolitis. A study was undertaken to investigate the time required for the ciliated epithelium to return to normal following bronchiolitis in infants treated with inhaled steroids or placebo. METHODS: Thirty one previously healthy term infants were studied as part of a clinical trial to determine the effect of 12 weeks of treatment with inhaled fluticasone (FP) or placebo via a spacer device (17 FP, 14 placebo). Nineteen healthy children aged 0-6 years previously studied in our department were used as controls. Nasal biopsy specimens were taken from infants with bronchiolitis and ciliary beat frequency (CBF) was measured before treatment and repeated 3, 6, 12, and 24 weeks later. The epithelial ultrastructure was examined by transmission electron microscopy and a normal errors mixed model based on normal controls was used to examine the time for cilia to return to normal in bronchiolitic infants. RESULTS: The mean CBF of infants with bronchiolitis (in Hz) at weeks 0, 3, 6, 12, and 24 were 0.5 (n = 4), 10.9 (n = 4), 12.0 (n = 9), 11.9 (n = 8), and 12.1 (n = 7) in the placebo group and 10.6 (n = 6), 11.4 (n = 9), 8.8 (n = 8), 10.9 (n = 4), and 13.2 (n = 7) in the FP group. The time for the epithelial ultrastructure to normalise was as follows: epithelial integrity score (13.1 weeks), % ciliated cells with loss of cilia (14.0 weeks), and % epithelial cells with abnormalities in projection (16.7 weeks) or mitochondria (15.9 weeks). Inhaled steroids had no significant effects on CBF or epithelial ultrastructure. CONCLUSION: Ciliary loss and epithelial abnormalities persist on average for 13-17 weeks following acute bronchiolitis in infancy.

Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults.

BACKGROUND: There are very few data on normal ciliary beat frequency, beat pattern, and ultrastructure in healthy children and adults. A study was undertaken to define ciliary structure, beat frequency and beat pattern in a healthy paediatric and young adult population. METHODS: Ciliated epithelial samples were obtained from 76 children and adult volunteers aged 6 months to 43 years by brushing the inferior nasal turbinate. Beating cilia were recorded using a digital high speed video camera which allowed analysis of ciliary beat pattern and beat frequency. Tissue was fixed for transmission electron microscopy. RESULTS: The mean ciliary beat frequency for the paediatric population (12.8 Hz (95% CI 12.3 to 13.3)) was higher than for the adult group (11.5 Hz (95% CI 10.3 to 12.7 Hz), p<0.01, t test); 10% (range 6-24%) of ciliated edges were found to have areas of dyskinetically beating cilia. All samples had evidence of mild epithelial damage. This reflected changes found in all measurements used for assessment of epithelial damage. Ciliary ultrastructural defects were found in less than 5% of cilia. CONCLUSION: Normal age related reference ranges have been established for ciliary structure and beat frequency. In a healthy population localised epithelial damage may be present causing areas of ciliary dyskinesia.

Haemophilus parainfluenzae infection of respiratory mucosa.

The pathogenicity of Haemophilus parainfluenzae (Hpi) in the respiratory tract is unclear, in contrast to the accepted pathogenicity of its close relative non-typable H. influenzae. We have investigated the interaction of two Hpi isolates with the mucosa of adenoid and bronchial tissue organ cultures. The adherence of bacteria to the mucosa of organ cultures, the effect of broth culture filtrates on human nasal epithelium, and interleukin (IL)-8 production by A549 cell cultures was investigated. Hpi 4846 adhered infrequently in clusters of pleomorphic cocco-bacilli to areas of epithelial damage, mucus and unciliated cells in adenoid organ culture experiments at 24 h, but not bronchial mucosa. Hpi 3698 was seen in only one adenoid and no bronchial organ cultures at 24 h. In separate experiments, Hpi 3698 was cleared more rapidly from the centre of the adenoid organ culture and was not cultured at 24 h. Although not adhering to the mucosa at 24 h, Hpi 3698, but not Hpi 4846, caused an increase in the amount of epithelial damage in both types of organ culture. Broth culture filtrates of both strains caused immediate slowing of ciliary beat frequency that progressed, and disrupted epithelial integrity. Dialysed culture filtrates of both strains stimulated IL-8 production by A549 cells, with the culture filtrate of Hpi 3698 being most potent. We conclude that two strains of Hpi varied in their adherence to adenoid tissue, and neither adhered to bronchial tissue. These results lead us to speculate that Hpi is only likely to be a pathogen in the lower respiratory tract when impaired airway defences delay bacterial clearance.

The effects of coronavirus on human nasal ciliated respiratory epithelium.

Human coronavirus (HCoV) accounts for 15-30% of common colds, but only one case report has described the effect of a coronavirus infection, that was asymptomatic, on human respiratory epithelium. The authors examined the effects of infection with HCoV on ciliary structure and function in healthy volunteers infected by intranasal inoculation with HCoV 229E. A further four volunteers were sham infected with ultraviolet-inactivated virus. Immediately before inoculation (day 0) and 3 days later (day 3), ciliated epithelium was obtained by brushing the inferior nasal turbinate. Ciliary beat frequency was determined and beat pattern analysed for evidence of dyskinesia (0=normal, 3=severely dyskinetic) using digital high-speed video photography. Ciliary ultrastructure was examined by transmission electron microscopy. Symptom diaries were kept for the duration of the study. All subjects inoculated with HCoV, including the three who did not develop symptoms of an upper respiratory tract infection, had disruption of their respiratory epithelium on day 3. Although there was no difference in the mean ciliary beat frequency between day 0 (11.3 Hz (95% confidence interval (CI): 8.6-14.0) and day 3 (9.4 Hz (95% CI 7.2-11.6)), there was a significant increase (p<0.05) in the ciliary dyskinesia score between day 0 (0.2 (95% CI 0-0.5)) and day 3 (1.1 (95% CI 0.5-1.7). In sham-infected subjects, no differences in epithelial integrity, or ciliary structure and function were found between day 0 and day 3. Inoculation of healthy volunteers with human coronavirus caused disruption of the ciliated epithelium and ciliary dyskinesia. This is likely to impair mucociliary clearance. Damage to the respiratory epithelium, due to human coronavirus infection, may occur without overt clinical symptoms.

Effect of pneumolysin on rat brain ciliary function: comparison of brain slices with cultured ependymal cells.

This study compares two models for examining ependymal ciliary function: rat brain slices cut from the fourth ventricle and primary ependymal cells in culture. The cilia from both preparations were very reproducible; each preparation had cilia beating at a constant frequency of between 38 and 44 Hz. With the brain slices, ciliary stasis occurred after 5 d in culture. However, ependymal cells had fully functional cilia for up to 48 d in culture. The pneumococcal toxin, pneumolysin, caused a dose-dependent inhibition of cilia beat frequency within 15 min in both models. There were no significant differences in the mean log 50% inhibitory concentration (pIC50) slice = 0.65 +/- 0.05, equivalent to 4.4 hemolytic units (HU)/mL; cells = 0.57 +/- 0.14, equivalent to 3.7 HU/mL. There were also no significant differences in the mean Hill slope factors for the curves (slice = 1.4 +/- 0.05; cells = 1.6 +/- 0.4). These data demonstrate that both models can be used to examine the acute (15-min) effects of pneumolysin on cilia beat frequency. The main advantage of the primary ependymal culture model is that considerably more cultured ependymal cells (approximately 70%) are available, compared with the number of ependymal cells on the brain slices (approximately 2%), thus reducing the number of animals used. A pure ependymal culture was not achieved (approximately 30% of the cells were not ciliated). The increased survival time of the ependymal cells compared with the brain slices make cultured ependymal cells more useful for examining long-term ciliary function, whereas brain slices may be more useful for examining the interactions between ependymal and other nearby cells.

Relative roles of pneumolysin and hydrogen peroxide from Streptococcus pneumoniae in inhibition of ependymal ciliary beat frequency.

Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H(2)O(2)) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 10(8) CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H(2)O(2) at a concentration as low as 100 microM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H(2)O(2) (10 microM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H(2)O(2) to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H(2)O(2), and in the presence of 10(8) CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H(2)O(2) release from the brain slice. Coincubation with catalase converted the H(2)O(2) produced by the pneumococci to H(2)O. Penicillin-induced lysis of bacteria dramatically reduced H(2)O(2) production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H(2)O(2), which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H(2)O(2) released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H(2)O(2)-induced cell toxicity, indicating a role for H(2)O(2) in the response. There is also a slight additive effect of pneumolysin and H(2)O(2) on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.

Respiratory and brain ependymal ciliary function.

The aims of this study were to compare beat frequencies of tracheal and ependymal cilia and the beat frequencies of ependymal cilia from infant and adult rats. The length of respiratory and ependymal cilia of infant and adult rats was also compared. We have developed an ex vivo model that allows ependymal and respiratory ciliary beat frequency to be measured with a high-speed video system. The beat frequencies of cilia, incubated at 37 degrees C, were measured after an incubation period of 30 min. Ependymal cilia beat at a similar frequency in 10- to 15-d-old rats (mean 38.8 Hz: 95% confidence intervals 37.1-40.6) as in adult animals (mean 40.7 Hz: 95% confidence intervals 38.5-42.9). However, respiratory cilia from adult animals beat (mean 20.9 Hz: 95% confidence intervals 14-27) at a significantly (p = 0.003) lower frequency than ependymal cilia. Ependymal cilia (mean length +/- SD: 8.2 +/- 0.3 microm) measured by scanning electron microscopy were significantly (p = 0.001) longer than respiratory cilia (5.5 +/- 0.6 microm) from the trachea of 9- to 15-d-old rats. Cilia did not grow longer between the time the rats were 9-15 d old and adulthood. Adult respiratory and ependymal ciliary length (mean +/- SD) were 5.6 +/- 0.5 microm and 8.1 +/- 0.2 microm, respectively. In summary, ependymal cilia beat at approximately twice the rate of respiratory cilia and are significantly longer.

Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth.

AIM: To examine strains of Pseudomonas aeruginosa for specific antifungal factors. METHODS: Two clinical strains of P aeruginosa with strong in vitro inhibition (by cross streak assay) of Candida albicans and Aspergillus fumigatus were examined. Both strains were isolated from sputum--one from a patient with cystic fibrosis and one from a patient with bronchiectasis. Bacterial extracts were fractionated by high performance liquid chromatography and examined by ultraviolet absorbance and mass spectroscopy. Antifungal activity against C albicans and A fumigatus was determined in a well plate assay. RESULTS: Pyocyanin was the major antifungal agent of P aeruginosa; 1-hydroxy-phenazine also possessed activity. Pyocyanin MICs for C albicans and A fumigatus were > 64 micrograms/ml. These phenazines were active against nine other yeast species pathogenic for man. Preliminary experiments also suggested possible inhibition of yeast mycelial transformation in C albicans by pyocyanin. CONCLUSIONS: There may be a role for pyocyanin and 1-hydroxyphenazine in the prevention of pulmonary candidiasis in patients colonised by P aeruginosa.

Effect of dirithromycin on Haemophilus influenzae infection of the respiratory mucosa.

Macrolides have properties other than their antibiotic action which may benefit patients with airway infections. We have investigated the effect of dirithromycin (0.125 to 8.0 microg/ml) on the interaction of Haemophilus influenzae with respiratory mucosa in vitro using human nasal epithelium, adenoid tissue, and bovine trachea. Dirithromycin did not affect the ciliary beat frequency of the nasal epithelium or the transport of mucus on bovine trachea, but dirithromycin (1 microg/ml) did reduce the slowing of the ciliary beat frequency and the damage to the nasal epithelium caused by H. influenzae broth culture filtrate. Amoxicillin (2 microg/ml) did not reduce the effects of the H. influenzae broth culture filtrate. H. influenzae infection of the organ cultures for 24 h caused mucosal damage and the loss of ciliated cells. Bacteria adhered to damaged epithelium and to a lesser extent to mucus and unciliated cells. Incubation of H. influenzae with dirithromycin at sub-MICs (0.125 and 0.5 microg/ml) prior to infection of the organ cultures did not reduce the mucosal damage caused by bacterial infection. By contrast, incubation of adenoid tissue with dirithromycin (0.125 to 1.0 microg/ml) for 4 h prior to assembling the organ culture reduced the mucosal damage caused by subsequent H. influenzae infection by as much as 50%. The number of bacteria adherent to the mucosa was reduced, although the tissue that had been incubated with dirithromycin (0.125 and 0.5 microg/ml) did not inhibit bacterial growth. This was achieved by a reduction in the amount of damaged epithelium to which H. influenzae adhered and a reduction in the density of bacteria adhering to mucus. We conclude that dirithromycin at concentrations achievable in vivo markedly reduces the mucosal damage caused by H. influenzae infection due to a cytoprotective effect.

Inhibition of calcium accumulation by the sarcoplasmic reticulum: a putative mechanism for the cardiotoxicity of adriamycin.

The aim of this study was to examine the effect of adriamycin (ADR) on calcium accumulation by the sarcoplasmic reticulum (SR). Chemical skinning of cultured rat myocardial cells compromised the barrier function of the cell membrane and thus permitted direct exposure of mitochondrial and non-mitochondrial sites to ADR. In the presence of ATP, and sodium azide, mitochondrial calcium accumulation was negligible. Furthermore, it has previously been shown that non-mitochondrial calcium accumulation is mediated mainly by the SR under these conditions. Incubation with 10 microM ADR for 2 hr reduced the level of calcium accumulation by the SR by 50%. A similar effect was obtained after 24 hr incubation with 1 microM ADR. The addition of ferric iron to the culture medium further reduced the level of calcium accumulation. Neither vitamin E nor beta-carotene affected calcium accumulation by the SR. These results suggest that ADR interferes with the calcium accumulation activity of the SR and that ferric iron potentiates this effect.

Effect of salmeterol on Pseudomonas aeruginosa infection of respiratory mucosa.

We have studied the effect of salmeterol on both P. aeruginosa interactions with the mucosa of nasal turbinate organ cultures and on pyocyanin-induced (20 microg/ml) and elastase-induced (100 microg/ml) damage to nasal epithelial cells. Organ cultures were exposed to salmeterol either by preincubation with 4 x 10(-7) M salmeterol for 30 min or by pipetting 20 microl of 4 x 10(-7) M salmeterol onto the organ culture surface immediately prior to bacterial inoculation. Infected organ cultures (8 h) had significantly (p < or = 0.01) increased epithelial damage, and P. aeruginosa was predominantly associated with damaged epithelium and mucus. Salmeterol significantly (p < or = 0.02) reduced epithelial damage caused by infection and the total number of adherent bacteria (p < or = 0.05), but bacterial distribution on the mucosa was unchanged. Nasal epithelial cells incubated with pyocyanin (20 microg/ml) or elastase (100 microg/ml) for 3 h had significantly (p < or = 0.05) increased cytoplasmic blebbing and mitochondrial damage versus control values. Elastase also significantly (p < or = 0.05) increased cell projection and reduced the level of ciliation. Cells preincubated with salmeterol (2 x 10(-7) M) showed a significant reduction in some features of cell damage caused by both toxins, which was inhibited by the beta2-adrenoceptor antagonist propranolol. Our results indicate that salmeterol reduces P. aeruginosa-induced damage to both organ culture and nasal epithelium.

Ciliary disorientation alone as a cause of primary ciliary dyskinesia syndrome.

Ciliary disorientation has been proposed as a variant of primary ciliary dyskinesia (PCD); cilia have normal ultrastructure and normal or near normal ciliary beat frequency (CBF) but lack efficacy because their beat direction is disorientated. We have identified 11 patients, including two siblings, with the clinical features of PCD, who satisfy these criteria. A chest radiograph, pulmonary function tests, nasal mucociliary clearance (NMCC), CBF, ciliary ultrastructure, and orientation were assessed in each subject. One patient had biopsies taken from the nose and both main bronchi. Eight patients had a computed tomography scan (CT) of the thorax; the clinical features were compatible with PCD. Cilia ultrastructure was normal and NMCC was absent in all cases. Mean CBF was normal (11.6-14.9 Hz) in five cases and slow in six (range 8.4-9.7 Hz). Ciliary beat pattern was stiff in seven cases, six of which had slow CBF. The cilia were disorientated when measured by both the central pair (range, 21.8 degrees - 26.4 degrees) and basal feet (range, 20.6 degrees - 28.9 degrees) compared with 16 normal controls (range, 11.0 degrees - 15.5 degrees and 12.3 degrees - 17.6 degrees, respectively). Two siblings had the clinical features of PCD and ciliary disorientation alone on repeated biopsies taken 10 yr apart. Orientation of cilia from the nose and bronchus was similar. Two cases had unchanged ciliary disorientation after 3 mo of treatment with antibiotics and topical corticosteroids. We concluded that ciliary disorientation alone can lead to the clinical syndrome of PCD.

The effects of low concentrations of antibiotics on epithelial damage caused by non-typable Haemophilus influenzae and bacterial morphology.

Sterile culture filtrates from non-typable Haemophilus influenzae (NTHi) grown in medium containing no antibiotics or 0.25 MIC of amoxycillin, ciprofloxacin or loracarbef were examined for their effect on the ciliary beat frequency (CBF) and structure of human respiratory epithelium. CBF slowing was significantly (P < 0.05) less with 0.25 MIC of all three antibiotics. The epithelium was significantly (P < 0.05) less disrupted with ciprofloxacin. The morphology of NTHi infecting human adenoid organ cultures after 24 h with or without 0.25 MIC of the same antibiotics was measured by scanning electron microscopy. Only ciprofloxacin caused a significant (P < 0.05) change in morphology.

Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

Interaction of fimbriated and nonfimbriated strains of unencapsulated Haemophilus influenzae with human respiratory tract mucus in vitro.

Adherence to mucus may influence bacterial colonization of the respiratory tract. Clinical isolates of nontypable Haemophilus influenzae (NTHi) from the respiratory tract are often fimbriated. We wondered whether fimbriated strains have a different adherence from related nonfimbriated strains. A microtitre plate assay has been developed to study adherence of nontypable H. influenzae to mucus. Wells were coated by incubation either with sol phase of sterile mucoid secretions or with purified preparations of mucins. Two laboratory pairs of fimbriated (F+) and nonfimbriated (F-) nontypable H. influenzae, and six fresh clinical isolates of fimbriated nontypable H. influenzae each with nonfimbriated partners derived by serial passage on agar, were cultured to mid-log phase, washed, and then added to the wells. They were then incubated at 37 degrees C for 30 min before washing to remove unbound bacteria. Adherent bacteria were desorbed by agitation with 0.5% Tween 80 and a viable count performed. The two fimbriated laboratory strains (n = 12 and n = 17), and 5 of the 6 fimbriated clinical isolates were more adherent to sol phase than their respective nonfimbriated partners. Two nonfimbriated clinical isolates were more adherent to plastic than their fimbriated partners. A fimbriated laboratory strain was more adherent than its nonfimbriated partner both to a purified preparation of high molecular mass mucin and to the glycopeptide fraction of the same. We conclude that fimbriated strains of nontypable H. influenzae have increased adherence to sol phase of mucus and purified human respiratory tract mucin. The interactions of fimbriae with mucus are likely to be complex, and may involve both nonspecific and specific interactions.