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 (pneumolysinnegative) 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 M 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 M) 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.
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.
In pathogenesis of celiac disease, the significance of prolamin peptide interactions with enterocytes is controversial. Changes in cellular metabolism induced by gliadin peptides, as well as uptake and presentation by enterocytes, are discussed. We analyzed peptide binding to enterocytic membranes as a potential key event. Binding capacities of brush border membranes isolated from small intestinal biopsies of untreated (n = 49) and treated celiac patients on a gluten-free diet (n = 30), as well as control subjects (n = 43), were measured with a dot blot chemiluminescence assay. Synthetic gliadin peptides comprising amino acid position 8-19 (G XIV) and 30-41 (G XI) of alpha-gliadins, a peptic-tryptic digest of gliadin (PT-GLI), and a synthetic zein peptide were used. Comparing treated celiac patients with controls, we observed significantly enhanced membrane-binding of PT-GLI [mean 122.4 densitometric units/microg (95% confidence interval 116.0-128.9) vs 108.9 (102.1-115.7)] and of zein peptide [50.2 (38.4-61.9) vs 28.8 (13.4-44.2)], but only slightly increased binding of the synthetic gliadin peptides G XIV [65.5 (60.6-70.5) vs 62.4 (56.3-68.5) and G XI [75.2 (69.8-80.6) vs 65.9 (55.2-76.5)]. Independent of patient group, membrane-binding capacities for celiac-active gliadin peptides exceeded those of the zein peptide. Thus, interaction of gliadin peptides with the apical enterocytic membrane was not found exclusively in celiac disease. Furthermore, increased binding capacities in treated celiac disease were not confined to celiac-active peptides. Quantitative differences in gliadin peptide binding as a primary characteristic in celiac disease might contribute to pathogenetic effects exerted on small intestinal epithelial cells.
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