Biomedical subjects
T Willems
Publications and source records attributed to T Willems.
IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells.
In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.
In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studies.
The aim of this study was to establish a collagen matrix-based nasal primary culture system for drug delivery studies. Nasal epithelial cells were cultured on derivatised (Cellagen membrane CD-24), polymerised (Vitrogen gel) and fibrillar (Vitrogen film) collagen substrata. Cell morphology was assessed by microscopy. The cells were further characterised by measurement of ciliary beat frequency (CBF), transepithelial resistance (TER), permeation of sodium fluorescein, mitochondrial dehydrogenase (MDH) activity and lactate dehydrogenase (LDH) release upon cell exposure to sodium tauro-24, 25 dihydrofusidate (STDHF). Among the three collagen substrata investigated, the best epithelial differentiated phenotype (monolayer with columnar/cuboidal morphology) occurred in cells grown on Cellagen membrane CD-24 between day 4 and day 11. Cell culture reproducibility was better with Cellagen membrane CD-24 (90%) in comparison with Vitrogen gel (70%) and Vitrogen film (< 10%). TER was higher in cells grown on Vitrogen gel than on Cellagen membrane CD-24 and Vitrogen film. The apparent permeability coefficient (Papp x 10(-7)cm s(-1)) of sodium fluorescein in these conditions was 0.45+/-0.08 (Vitrogen gel) and 1.91+/-0.00 (Cellagen membrane CD-24). Except for LDH release, CBF and cell viability were comparable for all the substrata. Based on MDH activity, LDH release, CBF, TER and permeation studies, Cellagen membrane CD-24- and Vitrogen gel-based cells were concluded to be functionally suitable for in-vitro nasal drug studies. Vitrogen film-based cultures may be limited to metabolism and cilio-toxicity studies.
Safety assessment of selected cyclodextrins - effect on ciliary activity using a human cell suspension culture model exhibiting in vitro ciliogenesis.
The objective of this study was to assess the cilio-inhibitory effect of a series of cyclodextrins using a human cell suspension culture system exhibiting in vitro ciliogenesis. Enzymatically released human nasal epithelial cells were cultured as sequential monolayer-suspension culture showing in vitro ciliogenesis. Ciliary beat frequency (CBF) was determined by computerized microscope photometry. Among the cyclodextrins investigated (gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, anionic-beta-cyclodextrin polymer, dimethyl-beta-cyclodextrin and alpha-cyclodextrin), it was shown that after 30 min of exposure, gamma-cyclodextrin (10% w/v), hydroxypropyl-beta-cyclodextrin (10.0% w/v) and anionic-beta-CD polymer (8.0% w/v) were not significantly cilio-inhibitory (P0.05). Similarly, CBF remained stable upon cell exposure to alpha-cyclodextrin (2.0% w/v) and dimethyl-beta-cyclodextrin (1.0% w/v). However, higher concentrations of alpha-cyclodextrin and dimethyl-beta-cyclodextrin resulted in mild to severe cilio-inhibition after 45 min of exposure. The effect of alpha-cyclodextrin (5.0% w/v; 54+/-4% cilio-inhibition) was partially reversible while dimethyl-beta-cyclodextrin (10% w/v; 36+/-4% cilio-inhibition) was irreversible. The cilio-inhibition observed in this model was lower than reported for chicken trachea model. Given the fact that (1) irreversible cilio-inhibition observed in this study occurred only at concentrations exceeding those used in pharmaceutical formulations and/or at an unusual exposure time (45 min) and that (2) in an in vivo situation, dilution and mucociliary clearance contribute to further decrease in local concentrations of the applied compound, the results of this study confirm the safety of the cyclodextrins investigated as nasal absorption enhancers.
Ciliary function analysis for the diagnosis of primary ciliary dyskinesia: advantages of ciliogenesis in culture.
The gold standard for the diagnosis of primary ciliary dyskinesia (PCD) is a dynein deficiency shown with transmission electron microscopy. However, there are many cases of PCD without dynein deficiency. When considering ciliary function, there are similar problems of sensitivity in diagnosis and there is also a major lack of specificity. Based on the normal ciliary function and ultrastructure and the absence of secondary abnormalities after ciliogenesis in sequential monolayer-suspension culture, the diagnostic value of ciliary function analysis after ciliogenesis was investigated in more than 70 PCD and 640 non-PCD cases. In biopsies, ciliary immotility was found in 66% of PCD cases but was also found in 8% of non-PCD cases. PCD was later confirmed in 61% of the biopsies with ciliary immotility. Normal ciliary beat frequency (CBF) was found in 20% of PCD biopsies. Coordinated ciliary activity was observed in 10% of PCD cases. After ciliogenesis in culture, ciliary immotility was present in 78% of the PCD cases but never in non-PCD cases. CBF was normal after ciliogenesis in 7% of the PCD cases and was always found in non-PCD cases. Absence of coordinated ciliary activity was found in 100% of PCD cases and 0% of non-PCD cases. In conclusion, while ciliary function analysis in a biopsy never proves, nor excludes the diagnosis of PCD, after ciliogenesis in culture CBF measurement can be diagnostic for PCD and reaches 100% specificity and sensitivity when considering coordinated ciliary activity, making it the single 100% diagnostic parameter for PCD.
Nasal nitric oxide.
Nitric oxide (NO) has witnessed an explosion of interest of scientists all over the world during the last decade. This small gaseous molecule is produced in many systems such as the nervous system, cardiovascular system, the upper and lower airways. In all of these it contributes to a number of (patho)physiological processes. Concerning the airways, NO concentrations in the upper respiratory tract are much higher (i.e. ranging from 200 to 2000 parts per billion (ppb)) than NO levels in the lower respiratory tract (i.e. ranging from 4 to 160 ppb). NO is most frequently measured using a chemiluminescence method, based on a reaction of NO with O3 resulting in the emission of light. In the airways NO exerts many functions in host defense, ciliary activity, inflammation and it is also an aerocrine messenger between the upper and lower airways. Nasal NO concentrations are influenced by age, physical exercise, smoking and certain drugs. Nasal NO is conveniently measured in all ages and can be used for screening of disease or monitoring the effects of treatment. Pathological conditions, as in allergic rhinitis, sinusitis, nasal polyps, cystic fibrosis and primary ciliary dyskinesia, result in altered nasal NO concentrations. The clinical relevance for measurement of nasal NO in different conditions, however, remains to be established.
Correlations between ciliary structure and ciliary function.
Mucociliary transport is a major defense mechanism of the airways. Mucociliary clearance is functionally and ultrastructurally organized at different levels from individual cilia to the ciliated tapestry. The correlations among ciliary beat frequency (CBF), secondary abnormalities (SCD), ciliary (dis)orientation (COR), coordinated ciliary activity and ciliary immotility were investigated based on the findings in over 700 non-PCD biopsies taken in the context of diagnostic investigations for respiratory problems. CBF decreased with increasing percentages of SCD, from 7.6 +/- 1.8 Hz (0-5% SCD) to 4.9 +/- 3.3 Hz (> 25% SCD). COR increased with increasing percentages of SCD, from 15 +/- 7 degrees for < 5% SCD to 28 +/- 8 degrees (> 25% SCD). SCD also correlated with ciliary (im)motility, but not with ciliary coordination. No correlation was found between COR and CBF. However, increased COR (28 +/- 8 degrees) was found in samples with only immotile cilia, compared to those with ciliary activity (19 +/- 9 degrees). Similar findings were demonstrated between COR and coordinated activity, particularly between immotile cilia (28 +/- 8 degrees) and those with coordinated ciliary activity (19 +/- 9 degrees). CBF values from samples with no coordinated activity (5.5 +/- 2.9 Hz) were significantly different from those with coordinated ciliary activity (7.4 +/- 1.6 Hz). In conclusion, mucociliary transport is a well organized, complex process with many interactions between parameters at various levels of functional and structural organization. SCD seems to play a crucial role. The correlations among the different parameters can help us further understand the functional and ultrastructural mechanisms needed for efficient mucociliary clearance.
Dynein arms and spokes after ciliogenesis in cultured respiratory epithelial cells from non-PCD individuals.
Dynein arms and spokes are crucial components of cilia. Reference values for the dynein arms and spokes were calculated based on biopsies from non-PCD patients as well as after ciliogenesis in culture. The mean values in the biopsies (n = 251) were 8.4 +/- 0.5, 2.9 +/- 0.7 and 4.7 +/- 1.0 for the outer dynein arms, inner dynein arms and spokes respectively. After ciliogenesis in culture (n = 462) identical values were found: 8.7 +/- 0.4, 3.0 +/- 0.4 and 5.5 +/- 0.6. The lower limits of normality can be set at 7.0 and 1.2 for the outer and inner dynein arms respectively. The dynein arms and spokes were not influenced by the percentage of secondary abnormalities. In conclusion, dynein arms and spokes are readily identified after ciliogenesis in culture. These parameters are independent of secondary ciliary dyskinesia.
Secondary ciliary dyskinesia is absent after ciliogenesis in culture.
Ultrastructural secondary ciliary dyskinesia (SCD) was measured using transmission electron microscopy in 301 biopsies and 439 samples after ciliogenesis in the sequential monolayer-suspension culture. Biopsies were taken in the context of exclusion of primary ciliary dyskinesia. SCD was frequently found in the biopsies: only 30% of the samples were normal (SCD < 5%), the mean percentage of SCD abnormalities was 11.9 +/- 12.9%. In 1/8 of the samples severe SCD (> 25%) was present. The most frequently encountered SCD abnormality was the membrane bleb, followed by the various peripheral microtubular abnormalities. With increasing total SCD the absence of the central pair became more important. After ciliogenesis in culture SCD was virtually absent: 1.0 +/- 1.8% for all 439 samples, 96% of the samples were within limits of normality (SCD < 5%). Moderate (15-25%) and severe SCD (> 25%) were never found. In more than 50% of the samples not one abnormality was found. There was no relation between the SCD in the biopsy and that after ciliogenesis. The absence of SCD after ciliogenesis is a major advantage for the diagnosis of PCD, specifically in cases with central pair abnormalities, peripheral microtubular pair abnormalities and those without a primary ultrastructural abnormality.
Ultrastructural expression of primary ciliary dyskinesia after ciliogenesis in culture.
During the period 1990-1999 84 PCD patients were identified and characterized. The expression of inherited abnormalities in primary ciliary dyskinesia after ciliogenesis was investigated in 41 patients with dynein deficiency, 6 patients with absence of the central pair of microtubules and 24 PCD patients with normal ultrastructure. In patients with dynein deficiency, the outer dynein arms counts were 1.9 +/- 1.0 in the biopsies and 1.6 +/- 0.7 after ciliogenesis. Secondary abnormalities were found in 15.8 +/- 20.4% of the transverse sections of cilia and only in 1.0 +/- 1.3% after ciliogenesis. Ciliary orientation was 28 +/- 11 degrees and 24 +/- 10 degrees respectively in biopsies and cultures. In patients with absence of the central pair this was found in 15 +/- 16% in biopsies and 21 +/- 19% after ciliogenesis. The values for the outer dynein arm were 8.4 +/- 0.3 and 8.7 +/- 0.2 and for the secondary abnormalities were 11.7 +/- 7.3% and 0.5 +/- 1.3% in the biopsies, respectively after ciliogenesis. In patients with normal ultrastructure the scores for the dynein arms were similar. Secondary abnormalities were found in 12.2 +/- 11.7% in the biopsies and 0.6 +/- 0.9% after ciliogenesis while ciliary orientation was respectively 21 +/- 7 degrees and 25 +/- 8 degrees. In conclusion, inherited abnormalities in primary ciliary dyskinesia are expressed after ciliogenesis, while secondary abnormalities are virtually absent, thereby facilitating the ultrastructural diagnosis.
Success rates of respiratory epithelial cell culture techniques with ciliogenesis for diagnosing primary ciliary dyskinesia.
The sequential monolayer-suspension culture technique has been used in over 800 samples during the period from January 1, 1990 till December 31, 1999. Patients were referred from all over Belgium. The culture technique was successful in 75% of the samples. In 85% of the patients a final conclusion regarding the exclusion/diagnosis of primary ciliary dyskinesia was made. In a total of 84 patients (10.3%) the final diagnosis was primary ciliary dyskinesia. Eighteen percent of the samples were considered normal, in 24% secondary ciliary dyskinesia was diagnosed.
Effects of pharmaceutical compounds on ciliary beating in human nasal epithelial cells: a comparative study of cell culture models.
PURPOSE: To test two in vitro human nasal epithelial cell culture systems for their ability to screen the effects of pharmaceutical compounds on ciliary beating. METHODS: Human nasal epithelial cells were cultured as monolayer and in a sequential monolayer-suspension culture with in vitro ciliogenesis. The influence of reference cilio-stimulatory compounds (glycocholate, isoprenaline), reference cilio-inhibitory compounds (chlorocresol, diphenhydramine) and pH on ciliary beating was investigated using computerized microscope photometry. RESULTS: Sodium glycocholate (0.5% w/v) maximally and reversibly increased CBF of the cells in both culture systems by 26 +/- 4% (monolayer) and 18 +/- 6% (suspension). Similarly, isoprenaline (10(-3) M) maximally, but irreversibly increased CBF of the cells by 14 +/-3% (monolayer) and 17 +/- 4% (suspension). Chlorocresol (0.005% w/ v) reversibly reduced the CBF of the cells by 50 +/- 6% (monolayer) and 34 +/- 4% (suspension); at a higher concentration (0.1% w/v) it resulted in instantaneous and irreversible ciliostasis. Diphenhydramine (0.1% w/v) reversibly reduced CBF in both culture systems by 45 +/- 13% (monolayer) and 69 +/- 5% (suspension); irreversible cilio-stasis occurred in less than 2 minutes in both culture systems upon cell exposure to diphenhydramine (1.0% w/v). In the monolayer culture system, CBF was stable only within the physiological pH range of 6.5-8.0; ciliary beating in the suspension culture remained stable within a pH range of 4.0-10.0. CONCLUSIONS: Both cell culture systems are suitable for screening the effects of pharmaceutical compounds on ciliary beating. Especially the sequential monolayer-suspension culture appears to be very promising as ciliary activity can be preserved for as long as 6 months.
Nasal ciliary beat frequency is age independent.
OBJECTIVES: Evaluate the age-dependency of ciliary beat frequency (CBF) in biopsies after ciliogenesis in culture. STUDY DESIGN: Retrospective analysis of CBF and ciliary ultrastructure in biopsies and after ciliogenesis from 203 individuals, aged 3 months to 74 years. METHODS: All patients with successful culture were included. Ciliogenesis was obtained using the sequential monolayer-suspension culture technique for dissociated nasal epithelial cells. CBF was measured using computerized microscope photometry. Secondary ultrastructural abnormalities were evaluated in transmission electron microscopy. RESULTS: There was no correlation between age and CBF, in either the biopsies (7.0 +/- 2.6 Hz; n = 113) or after ciliogenesis in culture (8.1 +/- 1.3 Hz; n = 203). Even in individuals older than 70 years, CBF was normal in bioptic material (6.7 +/- 1.7 Hz) and after ciliogenesis in culture (7.9 +/- 1.0 Hz). Also, in individuals less than 1 year of age CBF was normal in biopsies as well as after ciliogenesis. CBF correlated inversely with the percentage of secondary ultrastructural abnormalities in the biopsies as seen with transmission electron microscopy: 8.1 +/- 1.8 Hz when ciliary ultrastructure was normal and 3.5 +/- 3.3 Hz in cases of severe secondary ciliary dyskinesia. After ciliogenesis in culture, ciliary ultrastructure was always normal, as was CBF. CONCLUSION: CBF is age independent but correlates with secondary ultrastructural abnormalities. CBF after ciliogenesis in culture is the intrinsic one.
Lethal femoral-facial syndrome: a case with unusual manifestations.
The femoral-facial syndrome is a very rare syndrome of uncertain inheritance comprising hypoplastic femora, microretrognathia, and a peculiar facies. We report an additional observation detected by ultrasound at 25 weeks and diagnosed at birth. In addition to the malformations usually described in this syndrome, there were heterotopias of the brain, partial agenesis of the corpus callosum, bilobed lungs, intestinal malrotation, and vertebral segmentation defects.
Outcome after right middle lobe syndrome.
The long-term pulmonary consequences of right middle lobe syndrome (RMLS) in childhood are not known. Therefore, outcome was evaluated in 17 children with RMLS diagnosed in early childhood (mean age, 3.3 years; SD, 1.1 year). Mean age at follow-up was 10.1 years (SD, 2.6 years). RMLS was defined as atelectasis of the right middle lobe (RML) of at least 1 month's duration and visible on the lateral view of the chest radiograph as a wedge-shaped density extending from the hilum anteriorly and downward. Seventeen children without personal history of allergy or respiratory tract disease were studied as control group. Five of 17 study group children had ongoing respiratory problems: symptoms of asthma were present in 4 patients, and cylindrical bronchiectasis was present in one patient. Chest radiograph at follow-up was abnormal in six patients. Pulmonary function tests, including mean and SEM for vital capacity (VC) (82% of predicted +/- 7 vs 94% predicted +/- 3), FEV1 (77% of predicted +/- 12 vs 96% of predicted +/- 4) and their ratio (75 +/- 5 vs 85 +/- 3) were significantly lower in patients with ongoing respiratory symptoms than in the control children. The provocative dose causing a 20% decrease in FEV1 (PD20) of methacholine was significantly lower in patients with ongoing symptoms at follow-up than in control children and in patients without symptoms at follow-up (2.8[2.2 to 3.1] vs 4.5[2.2 to 8.8] and 9.2[2.3 to 24] mg/mL; median and P25-75, p < 0.05). Age at initial diagnosis tended to be younger in patients with ongoing symptoms at follow-up (2.3 +/- 0.7 years vs 3.8 +/- 0.4 years; p < 0.08).
Nucleolar organizer regions in the normal and carcinomatous epithelium of the uterine cervix. A morphometric study.
Nonhistone nucleoproteins associated with the nucleolar organizer regions (NORs), the genes coding for the ribosomal RNA precursor, can be visualized by silver staining. The black dots (AgNORs) appearing on the nuclei are thought to reflect cell differentiation. In this study, AgNORs were counted and their area was measured and compared with the area of the nuclei in normal and carcinomatous cells of the uterine cervix. The number of AgNORs per nucleus was significantly higher in the endocervical than in the basal exocervical epithelium (p less than 0.005) and in the carcinomatous epithelium, either in situ or invasive, than in both normal epithelia (p less than 0.002). Individual AgNORs were significantly smaller in carcinoma in situ than in endocervical epithelium (p less than 0.05) or in invasive carcinoma (p less than 0.01). Significant differences were also found in the total AgNORs area per nucleus between the following groups: basal exocervical versus endocervical epithelium (p less than 0.01), basal exocervical and endocervical epithelium versus invasive carcinoma (p less than 0.001), and in-situ versus invasive carcinoma (p = 0.02). The conclusions are that the number and the total area of AgNORs per nucleus increase with the differentiation of the cell or with its carcinomatous transformation, but no prognostic significance can be drawn so far from our measurements.
Effect on ovulation of surgically induced endometriosis in rabbits.
To study the effect of endometriosis on follicular rupture, endometrial tissue was autografted to New Zealand White rabbits. Endometrium was surgically implanted into the peritoneal cavity or into the rectus muscle. Human chorionic gonadotropin was administered to induce ovulation. During three subsequent laparotomies, the number of corpora lutea and stigmata were counted. The viability of the implants was demonstrated histologically. Ovaries were removed during the last laparotomy and ovarian serial sections were examined. In rabbits with peritoneal induced endometriosis, the percentage of stigmata/corpora lutea was significantly decreased. Macroscopic study was confirmed by histological examination. Indeed, a high incidence of entrapped oocytes was found in rabbits with peritoneal endometriosis. Extraperitoneal endometriosis had no effect on ovulation. Our data demonstrated that endometriosis induced a failure of follicular rupture. After endometriumectomy, no failure to ovulate was observed, suggesting that the effect of endometriosis on the ovulation disappeared with excision of endometrial implants.
[Unruptured luteinized follicle syndrome and experimental endometriosis in the female rabbit].
To study the effect of endometriosis on follicular rupture, endometrial tissue was autografted to New Zealand White rabbits. Endometrium was surgically implanted into the peritoneal cavity. Human chorionic gonadotropin was administered to induce ovulation. The viability of the implants was demonstrated histologically during three subsequent laparotomies, the number of corpora lutea and stigmata were counted. Ovaries were then removed and ovarian serial sections were examined. In rabbits with peritoneal induced endometriosis, the percentage of stigmata per corpora lutea was significantly decreased. Macroscopic study was confirmed by histological examination. Indeed, a high incidence of entrapped oocytes was found in rabbits with peritoneal endometriosis. Our data demonstrated that endometriosis induced a failure of follicular rupture.