B.L. Grossbard

Hepatic oval cells (HOC) are thought to be a type of facultative stem cell that arises as a result of certain forms of hepatic injury. A new and more efficient model has been established to activate the oval cell compartment in mice by incorporating 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) in a standard chow at a concentration of 0.1%. At the present time, very few markers exist for the mouse oval cells. One accepted marker is A6, an uncharacterized epitope recognized by mouse hepatic oval cells and it is accepted to be an oval cell marker. Sca-1 is a cell surface marker used to identify hematopoietic stem cells in conjunction with Thy-1+, CD34+, and lineage-specific markers. Both the CD34 and Sca-1 antigens are not normally expressed in adult liver, but are expressed in fetal liver, presumably on the hematopoietic cells. We report herein that mouse oval cells express high levels of Sca-1 and CD34, as well as CD45 surface proteins. Immunohistochemistry revealed that the cells expressing Sca-1/CD34/CD45 were indeed oval cells because they co-expressed the oval cell-specific marker A6 (94.57% +/- 0.033%), as well as alpha-fetoprotein (AFP) (75.92% +/- 0.071%). By using Sca-1 antibody in conjunction with magnetic activated cell sorting (MACS), followed with a flow cytometric cell sorting (FACS) method for CD34 and CD45, we have developed a rapid oval cell isolation protocol with high yields of greater than 90%. In conclusion, we have an efficient murine model for the production and isolation of large numbers of highly purified oval cells. Our system works with most strains of mouse, which will facilitate both in vivo and in vitro studies of mouse hepatic oval cells.

OBJECTIVE: To: (1) determine the success of medical infrared imaging (MII) in identifying dogs with TLIVDD, (2) compare MII localization with magnetic resonance imaging (MRI) results and surgical findings, and (3) determine if the MII pattern returns to that of normal dogs 10 weeks after decompression surgery. STUDY DESIGN: Prospective case series. ANIMALS: Chondrodystrophic dogs (n = 58) with Type I TLIVDD and 14 chondrodystrophic dogs with no evidence of TLIVDD. METHODS: Complete neurologic examination, MII, and MRI studies were performed on all dogs. Dogs with type I TLIVDD had decompressive surgery and follow-up MII was performed at 10 weeks. Pattern analysis software was used to differentiate between clinical and control dogs, and statistical analysis using anatomic regions of interest on the dorsal views were used to determine lesion location. Recheck MII results were compared with control and pre-surgical images. RESULTS: Computer recognition pattern analysis was 90% successful in differentiating normal dogs from dogs affected by TLIVDD and 97% successful in identifying the abnormal intervertebral disc space in dogs with TLIVDD. Statistical comparisons of the ROI mean temperature were unable to determine the location of the disc herniation. Recheck MII patterns did not normalize and more closely resembled the clinical group. CONCLUSIONS: MII was 90% successful differentiating between normal dogs and 97% successful in identifying the abnormal intervertebral disc space in dogs with TLIVDD. Abnormal intervertebral disc space localization using ROI mean temperature analysis was not successful. MII patterns 10 weeks after surgery do not normalize.

Similar to Streptococcus mutans, buffer suspensions of Lactobacillus casei, Lactobacillus plantarum, and Fusobacterium nucleatum all undergo cell lysis when treated with the lysozyme-protease-inorganic monovalent anion antibacterial system. For Lactobacillus species treated with lysozyme and proteases at pHs of 4 and 5.3, lysis resulted when a lytic activating concentration of bicarbonate anion followed enzyme treatment. Furthermore, synergistic lysis of these bacteria was noted when lysozyme-protease treatment was followed by bicarbonate anion used in combination with chloride or fluoride anions. Noteworthy, the halides were not active in promoting lysis when used by themselves in the absence of bicarbonate. For F. nucleatum suspended at pH 6.9, lysis was dependent upon the ionic strength of the buffer and resulted when lysozyme-protease treatment of the organism was followed by 100 mmol/L bicarbonate activation. When lysozyme and proteases were omitted from the incubation mixtures and replaced by stimulated whole saliva, pH 5.3, lysis was observed only with L. plantarum and S. mutans, but not with L. casei. The latter could be lysed, however, if suspended in saliva which was diluted several-fold with distilled water. In experiments where lysozyme was selectively depleted from whole saliva by immunoadsorption affinity chromatography, the great majority of the lysis capability of the saliva for L. plantarum was lost, although a significant degree of lysis appeared to be due to salivary factors other than lysozyme. F. nucleatum was also found to lyse in saliva at neutral pH, suggesting that both Gram-positive and Gram-negative oral bacteria may be susceptible to this antibacterial system in vivo.

The ability of lysozyme to aggregate and lyse the gram-negative capnophilic periodontal microorganism Capnocytophaga gingivalis 2010 was monitored optically at 540 nm. Both hen egg white and chromatographically purified human lysozymes had significant but similar aggregation potentials for both logarithmic- and stationary-phase bacteria. In general, an increase in enzyme concentration resulted in a graded increase in both the initial and maximum changes in turbidity which occurred during the reaction period. The greatest change in turbidity occurred within the initial minutes of interaction of lysozyme and the cells, and the extent of aggregation paralleled a rapid depletion of lysozyme by the suspensions during the first minute of its incubation with the bacteria. Interestingly, the muramidase inhibitors N-acetyl-D-glucosamine and histamine did not block aggregation, whereas maleylation of lysozyme completely inhibited its aggregating ability. Demaleylation, however, restored aggregation activity comparable to the native enzyme, indicating that maleylated lysozyme retained its integrity and that aggregation was primarily dependent on charge. The addition of up to physiological concentrations of NaHCO3 and NaCl to cell aggregates resulted in varying degrees of deaggregation and lysis. Surprisingly, ultrastructural analysis of lysozyme-treated cells revealed morphological changes with or without the addition of salt. Damage appeared to occur at the blunted polar end of the cells where there was a large spherical outpouching bordered by a damaged cell envelope. Damaged cells uniformly contained dense granular cytoplasmic debris. In effect, the cationic enzyme lysed C. gingivalis 2010, which was not apparent in the spectrophotometric assay. The paradoxical finding that during bacterial aggregation there was lysis may be of significance to the further elucidation of lysozyme's antibacterial role in the gingival sulcus.

The specificity of lysozyme determinations in human parotid and submandibular-sublingual salivas of two subjects was assessed by comparison of lysozyme concentrations in native acidified salivas with purified enzyme obtained by immunoadsorbent fractionation of the salivas. Lysozyme concentrations were measured by the turbidimetric catalytic method and by a newly developed enzyme-linked immunosorbent assay (ELISA). The validity of the assays was established by comparing assay results with enzyme concentration values determined from optical density-extinction coefficient calculations of the purified lysozyme peak. Values for purified enzyme were found to be similar, irrespective of the assay used to determine lysozyme concentrations, and were in agreement with extinction coefficient calculations. Based on the ELISA technique, recoveries of lysozyme from both parotid and submandibular-sublingual salivas were greater than 75 and 90%, respectively. Similar recoveries were noted for parotid saliva when determinations were based on the turbidimetric assay. However, the ELISA and turbidimetric assays differed with respect to lysozyme levels in submandibular-sublingual saliva because of the apparent presence of an enhancement factor which gave rise to higher lysozyme values in the catalytic assay and therefore resulted in low recoveries of purified enzyme. This catalytic enhancement factor was present in the nonadsorbed fraction of both subjects, as higher lysozyme activities were noted when nonadsorbed fractions were added to affinity-purified lysozymes. Lysozyme levels were also determined in the parotid and submandibular-sublingual salivas of caries-resistant and -susceptible adults. In general, levels of lysozyme in parotid saliva were lower in comparison to submandibular -sublingual saliva; however, significant differences in enzyme concentration were not evident between the caries-resistant and caries-susceptible subjects.