Mucins in the host defence against Naegleria fowleri and mucinolytic activity as a possible means of evasion.

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM). This parasite invades its host by penetrating the olfactory mucosa. During the initial stages of infection, the host response is initiated by the secretion of mucus that traps the trophozoites. Despite this response, some trophozoites are able to reach, adhere to and penetrate the epithelium. In the present work, we evaluated the effect of mucins on amoebic adherence and cytotoxicity to Madin-Darby canine kidney (MDCK) cells and the MUC5AC-inducing cell line NCI-H292. We showed that mucins inhibited the adhesion of amoebae to both cell lines; however, this inhibition was overcome in a time-dependent manner. N. fowleri re-established the capacity to adhere faster than N. gruberi. Moreover, mucins reduced the cytotoxicity to target cells and the progression of the illness in mice. In addition, we demonstrated mucinolytic activity in both Naegleria strains and identified a 37 kDa protein with mucinolytic activity. The activity of this protein was inhibited by cysteine protease inhibitors. Based on these results, we suggest that mucus, including its major mucin component, may act as an effective protective barrier that prevents most cases of PAM; however, when the number of amoebae is sufficient to overwhelm the innate immune response, the parasites may evade the mucus by degrading mucins via a proteolytic mechanism.

Human salivary mucin MUC7 12-mer-L and 12-mer-D peptides: antifungal activity in saliva, enhancement of activity with protease inhibitor cocktail or EDTA, and cytotoxicity to human cells.

MUC7 12-mer-L exhibits potent in vitro antifungal activity in low-ionic-strength buffers. In this study, we investigated the anticandidal activity and stability of MUC7 12-mer-L and its all-D-amino-acid isomer, along with Hsn5 12-mer (P113) and magainin-II, in human clarified and unclarified saliva in the absence or presence of protease inhibitor cocktail (PIC, which includes EDTA) or EDTA alone. In the absence of PIC or EDTA in saliva, only MUC7 peptides showed significant candidacidal activity. At a 100 microM concentration in clarified saliva and unclarified saliva, MUC7 12-mer-D demonstrated 94 versus 64% killing, respectively; MUC7 12-mer-L showed 57 versus 32% killing; Hsn5 12-mer showed 16 versus 0% killing; and magainin-II showed no killing. Addition of PIC or EDTA to either saliva caused the enhancement of antifungal activities of all peptides, although to different degrees. Taken together, the results suggest that EDTA (a metal-dependent protease inhibitor and/or divalent cation chelator) enhanced the antifungal activity of all four peptides mainly by chelation of divalent cations present in saliva (known to inhibit peptide antifungal activity), and PIC enhanced the activity of the three L peptides above that achievable by EDTA alone through inhibition of all classes of proteases. Peptide stability in saliva monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no degradation of MUC7 12-mer-D and 23, 60, and 75% degradation of MUC7 12-mer-L, Hsn5 12-mer, and magainin-II, respectively. Cytotoxicity assays determined that, at 100 microM peptide concentrations, MUC7 12-mer-D and 12-mer-L caused 3.5 and 4.3% hemolysis in phosphate-buffered saline and no toxicity to the HOK-16B cell line (derived from normal human oral keratinocytes). In summary, MUC7 12-mer peptides appear to be excellent candidates for investigation of antifungal activity in in vivo models of oral candidiasis.

Assessment of glycosylation-dependent cell adhesion molecule 1 as a correlate of allergen-stimulated lymph node activation.

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose-response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.

Further characterization of monoclonal antibodies to lipopolysaccharide of Salmonella Minnesota strain R595.

We have shown here that despite the use of monoclonal antibodies with well-defined epitope-specificities, and despite testing them in the most simple animal model available (i.e., mixing of homologous LPS with Mab prior to injection), we are not yet able to explain why some of the antibodies were effective and others not. For some of the clones (e.g., clone 20), an even better definition of binding sites is currently taking place in an attempt to obtain this understanding. We also do not yet understand why clone 20 was not effective in the mucin model, while using much lower amounts of injected antibody, and much higher challenge doses, this Mab was effective against E. coli in the gentamicin-treated mouse model. Very clear is, however, that in order to be protective in the latter model, Mabs are not required to be specific for lipid A. In the future it will be essential to develop procedures which measure specific interaction between smooth LPS/bacteria and antibodies to the LPS core region. In addition, it will be of great help when the chemical structure of non-substituted, rough-form LPS, as occurring in smooth LPS preparations, would be defined. This applies also to O-substituted core molecules.

Inactivated Saccharomyces cerevisiae as virulence-enhancing agent in the toxicity test of Salmonella typhi and the immunity assay of typhoid vaccine in mice.

Mucin has been used as virulence-enhancing (VE) agent in estimation of the toxicity of bacteria to mice and the potency of bacterial vaccines such as typhoid vaccine. Discontinuance of the acceptable brand of mucin made it necessary to search for an effective alternative. Five percent inactivated Saccharomyces cerevisiae (IY) showed a significant higher VE effect on S. typhi in mice (132 colony forming units/LD50) with lower viscosity when compared to 5% mucin (740 colony forming units/LD50), and either one could promote bacterial lethal toxicity in mice more than 10(4) times. The strong correlation (r2 = 0.9765) between the immune dose and the ratio of mouse death was found by using 5% IY as VE agent in the active mouse protection test for typhoid vaccine. The relative potencies of a typhoid vaccine to the reference vaccine obtained by using 5% mucin or 5% IY as VE agent were not significant difference. Therefore, IY was a suitable substitute for mucin as a VE agent in the toxicity test of bacteria and in the immunity assay of typhoid vaccines.

Prevention of the enhancing effect of mucin and iron in mouse meningococcal infection.

In vivo resistance of mice to Neisseria meningitidis was entirely abrogated by a concomitant administration of mucin and iron with N. meningitidis organisms. Resistance, however, was restored when the latter challenge was given to animals which had been immunized 7 days previously with a crude extract of meningococcal antigens (MA), BCG, or proteose peptone. These results suggest that depression or activation of the reticuloendothelial system (RES) may be important in resistant of mice to meningococcal infection. Also, like BCG, MA inoculation was able to prevent infection by Listeria monocytogenes indicating its marked ability to activate the RES. The data show that immunization can induce nonspecific RES stimulation and that the nonspecific resistance persists for at least 7 days.

Dry coating of intraocular lenses with bovine submaxillary mucin.

Uncoated intraocular lenses caused extensive corneal endothelial cell damage after in vitro contact (mean, 30.9% cell loss), whereas lenses coated with bovine submaxillary mucin type 1 caused minimal cell loss (mean 4.3%). No observable in vivo toxicity in the eye occurred when 0.1 ml of 3, 6, or 9% solution was injected into the anterior chamber. Mucin-coated lenses can be sterilized by ethylene oxide with no measurable adsorbence into the coating. Scanning electron microscopy confirmed the results from vital staining.