Expression of the sialosyl-Tn epitope on CD45 derived from activated peripheral blood T cells.

The cell surface protein tyrosine phosphatase CD45 is a major target of IgM anti-T cell autoantibodies in systemic lupus erythematosus (SLE). The autoreactive determinants on CD45 are O-linked glycans expressed on activated T cells and certain T cell lines, rather than linear or conformational polypeptide epitopes or N-linked glycans. To identify oligosaccharide structures that may play a role in the functional interactions of CD45 or are candidate target epitopes of SLE anti-CD45 autoantibodies, autoreactive CD45 purified from Jurkat T cells and non-autoreactive CD45 purified from CLL B cells were tested by ELISA for expression of mucin-type O-glycan structures. Monoclonal antibodies (mAbs) directed against blood group A, type 1 H chains, type 2 H chains, T, Le(a), sialylated-Le(a), Le(b), sialylated-Le(c), Le(x), sialylated-Le(x), multi-fucosylated Le(x), Le(y), and sialylated-extended Le(v) failed to react with CD45 from either B cells or T cells. However, mAbs directed against Tn (galNAcalpha1-->O-ser/thr) or sialosyl-Tn (neuNAcalpha2-6gaINAcalpha1-->O-ser/thr) structures reacted with CD45 derived from Jurkat T cells, but not from CLL B cells. Anti-Tn mAbs also reacted in western blotting procedures with CD45 isolated from Jurkat T cells, but did not react with CD45 isolated from CEM, MOLT-3, or PEER T cells; Daudi, Raji, or CLL B cells; or resting or Con A-activated PBL. However, anti-sialosyl-Tn mAbs stained blots of CD45 isolated from Jurkat and CEM T cells and Con A-activated PBL, a pattern of reactivity similar to that of the anti-CD45 autoantibodies. Flow cytometric analyses demonstrated that the sialosyl-Tn epitopes are expressed on a subpopulation of CD4 +/CD8- T cells.

Purified streptococcal cell wall (PG-APS) causes experimental otitis media.

Purified peptidoglycan-polysaccharide from Group A Streptococcus pyogenes cell wall (PG-APS) was prepared under sterile, pyrogen-free conditions and injected percutaneously into the middle ear cavities (MEC) of groups of gerbils. Each group was observed otoscopically at 1, 2, 5, 7, 9, 12 and 14 days. Histologic specimens were obtained from each group at 2, 5, 7, 9 and 14 days. Histologically, PG-APS produced mild, acute inflammation during the first 3 days and mild to moderate inflammation with features of both acute and chronic pathology from days 5 through 9. An early response by plasma cells and macrophages was seen. Most animals had recovered with minor sequelae by day 14. Challenge intraperitoneally with 20 micrograms of PG-APS at 14 days following MEC injection of PG-APS did not exacerbate the middle ear inflammation. Five chinchillas, tested for comparison, responded similarly to PG-APS.

Experimental otitis media with Moraxella (Branhamella) catarrhalis.

Two hundred fifty gerbils and 7 chinchillas were utilized in 11 experiments to determine the effect of inoculating viable and heat-killed suspensions of Moraxella (Branhamella) catarrhalis into the middle ear cavity. Development of otitis media was observed by otoscopy and histopathology. Gerbils were found to be susceptible to 2 x 10(4) viable M catarrhalis cells. Depending on the number of cells inoculated, the resulting untreated inflammation was a very mild to moderately severe, self-limiting disease with no permanent sequelae except in animals inoculated with high [10(6) to 10(7)] numbers of bacteria. Viable bacteria could not be isolated from the middle ears 24 hours after inflammation was induced. Heat-killed cells produced less severe acute inflammation with no permanent sequelae. We conclude 1) gerbils and chinchillas are susceptible to a self-limited inflammation caused by M catarrhalis, 2) no infection occurs, since viable bacteria cannot be recovered from middle ear aspirates, and 3) viable cells produce more severe inflammation than heat-killed cells.

Cecal and fecal bacterial flora of the Mongolian gerbil and the chinchilla.

The Mongolian gerbil is being increasingly used as a laboratory animal and as a pet. Both chinchillas and gerbils are used as animal models for otitis media and other otic research. Previously, only incomplete information was available regarding the indigenous bacterial flora of the lower intestinal tracts of these coprophagic animals. Using the strict anaerobic methodology of the Virginia Polytechnic Institute Anaerobe Laboratory, we studied the predominant bacterial flora of the cecum and fecal pellets of the gerbil and the chinchilla and the bacterial flora of digesta pellets in the proximal colon. We found species of the following anaerobic genera in high dilutions of gerbil fecal pellets: Bifidobacterium, Clostridium, Propionibacterium, Lactobacillus, and Bacteroides. Only lactobacilli were found in high dilutions of digesta from the upper colon, although the cecum yielded Peptostreptococcus, Bifidobacterium, Clostridium, Lactobacillus, Propionibacterium, and Bacteroides species from high dilutions of cecal contents. The facultatively anaerobic and aerobic flora isolated consisted of species of Bacillus, Streptococcus, Staphylococcus, Acinetobacter, Alcaligenes, Escherichia, Pasteurella, and Pseudomonas plus several unidentifiable organisms. Species of Bifidobacterium, Bacteroides, Eubacterium, and anaerobic Lactobacillus were isolated from chinchillas.

Mongolian gerbil tympanic membrane. Normal and with induced otitis media.

Otoscopic observation of the tympanic membrane is the most effective noninvasive means of diagnosing and following the course of experimental otitis media in animal models of the disease in both short-term and in longitudinal studies. Because of the difficulty of viewing the entire tympanic membrane of the gerbil model of otitis media without manipulation, we present methods of visualizing and color illustrations of the normal tympanic membrane of the Mongolian gerbil, Meriones unguiculatus. These are compared with illustrations of animals with acute experimental otitis media. An artist's rendition of the entire normal gerbil tympanic membrane is presented. These methods and illustrations will enable investigators to more rapidly and easily adopt the Mongolian gerbil as an animal model for studies of otitis media.

Bacterial flora in spontaneously occurring aural cholesteatomas in Mongolian gerbils.

Bacteria were isolated from 29 Mongolian gerbils, Meriones unguiculatus, with spontaneous aural cholesteatomas. We identified 148 cultures, 44 from the middle ear surface of cholesteatomas and 104 from cholesteatoma contents. We could only identify 63 cultures to the genus level, although we identified 85 cultures as belonging to 21 different species. We found on the surfaces of cholesteatomas representatives of 9 genera, from which 8 species could be identified, and representatives of 19 genera within the cholesteatoma sac, from which 21 species could be identified. The most common bacterial genera isolated were Staphylococcus, Pseudomonas, and Corynebacterium among the aerobic genera, and Bacteroides and Peptococcus among the anaerobic genera. The bacterial flora of gerbilline cholesteatomas was found to be diverse, resembling the flora found to be associated with human cholesteatomas. The flora also resembled the organisms found transitorily within the normal middle ear cavity of gerbils, except for a higher incidence of pseudomonads.

Idiopathic hyperaldosteronism in a dog.

Idiopathic hyperaldosteronism was diagnosed in an 8-year-old castrated male Yorkshire Terrier, based on increased concentration of plasma aldosterone, hypertension, hypernatremia, decreased natriuresis, hypokalemia, and hyperkaluria. Unilateral adrenalectomy was performed after visualization of a nodule on the right adrenal gland. Hyperplasia of the zona glomerulosa and increased postoperative aldosterone concentrations supported the diagnosis of idiopathic hyperaldosteronism.

Experimental pneumococcal otitis media: longitudinal studies in the gerbil model.

In order to ascertain that the Mongolian gerbil, Meriones unguiculatus, is an acceptable model for studying the development of chronic from acute otitis media, we used previously published methods for experimental otitis media in a longitudinal study of the acute disease and sequellae. The gerbil was found to be susceptible to as few as 30 viable Streptococcus pneumoniae type 3 cells, indicating that only a few viable pathogens are able to cause the disease. Untreated experimental infections with S. pneumoniae type 23 resulted in a mild, self-limiting disease with little permanent sequellae, while S. pneumoniae type 3 produced severe disease characterized by an acute phase of from 2 to 3 weeks, followed by the development of new bone formation and a vascularized granulation tissue which persisted throughout the 13-week study. Viable pneumococci could be recovered from the middle ears for only two weeks. We conclude that the gerbil is a useful model for otitis media.

Longitudinal studies of experimental otitis media with Haemophilus influenzae in the gerbil.

Haemophilus influenzae non-typable strain 119 was found to cause severe otitis media with sequellae when inoculated into the middle ear cavities of the Mongolian gerbil, Meriones unguiculatus. Acute inflammation was followed by the development of highly vascular granulation tissue and formation of new bone within the middle ear bulla. These changes persisted throughout the 14-week study in the untreated animals. The gerbil was variably susceptible to otitis media caused by inoculating 30-3000 bacterial cells and 100% susceptible to greater than 3000 cells. The susceptibility of the gerbil to common etiological agents of otitis media allows its use as an appropriate model of the disease.

Superoxide dismutase in ruminal bacteria.

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.

Bactericidal activity of wet cerumen.

The viable populations of seven species of bacteria were reduced 17% to 99% by treatment with a 3% suspension of human cerumen of the soft or "wet" type. Species tested for susceptibility to cerumen were Staphylococcus aureus, Staphlylococcus epidermidis, Streptococcus pyogenes, Propionibacterium acnes, Corynebacterium spp, Escherichia coli, and Serratia marcescens. The reduction depended upon the species of bacterium and the age of the culture. Pathogenic species appeared to be more susceptible than others, while cultures in logarithmic growth were more susceptible than stationary phase cultures to the bactericidal activity of wet cerumen. There appeared to be little difference in the bactericidal activity between the two lots of cerumen used. The bactericidal activity of wet cerumen was found to be quite similar to that of dry cerumen as reported earlier in the literature.

Experimental otitis media in gerbils and chinchillas with Streptococcus pneumoniae, Haemophilus influenzae, and other aerobic and anaerobic bacteria.

To ascertain the usefulness of Mongolian gerbils as an inbred model for otitis media, 52 Mongolian gerbils (Meriones unguiculatus, strain MONT/Tum) were compared with 26 chinchillas (Chinchilla laniger) for susceptibility to Streptococcus pneumoniae type 3. Haemophilus influenzae type b, and a polymicrobic culture including anaerobes (Streptococcus intermedius, Propionibacterium acnes, Staphylococcus epidermidis, and Corynebacterium sp.). Organisms were inoculated percutaneously into the superior chamber of the middle ear bulla. The gerbils and chinchillas shared similar susceptibilities and responses to the inoculated organisms as determined by X-ray, otoscopic, histopathological, and microbiological determinations at 5 to 7 days. Koch's postulate studies proved the role of S. pneumoniae and H. influenzae in the pathology found in both animal models. The animals were also susceptible to the polymicrobic culture, although the relative virulence of the individual members of this mixture was low, suggesting that these species potentiated as a polymicrobic mixture. The Corynebacterium sp. appeared to elicit the greatest histopathological response in chronic (8-week) studies in gerbils. The gerbils were found to be useful as an alternative animal model for the study of otitis media of bacterial etiology.

Comparative anatomy of eustachian tube and middle ear cavity in animal models for otitis media.

The comparative anatomy of the normal eustachian tube (ET) and normal middle ear cavity of three animal species (rat, gerbil, and chinchilla) is described relating to the usefulness of these animals as models for otitis media (OM). Routine histological and anatomical techniques and procedures were used. The gerbil and chinchilla, although of different sizes, are quite similar, having hypertrophied middle ear bullae, nearly vertical ET, and similar histology including seromucous glands draining directly into the ET. In contrast, the rat has a small bulla, a nearly horizontal ET, and a large concentration of goblet cells but few mucous glands in the ET. It appears that the chinchilla and the gerbil may serve as alternative models for OM research. Chinchillas and gerbils are relatively free of naturally occurring OM, while the rat has a high incidence of naturally occurring OM.

Prophylactic cefazolin in gastric bypass surgery.

Because surgery in obese patients is associated with a high risk of infection, gastric bypass procedures offer an excellent opportunity to test the efficacy of prophylactic antibiotics. Accordingly, a double-blind prospective trial of prophylactic cefazolin was carried out in 53 consecutive patients who underwent gastric bypass surgery for morbid obesity. The patients were randomized to two treatment arms: (1) cefazolin intravenously, 1 gm 2 hours prior to surgery, at induction of anesthesia, and then 0.5 gm every 6 hours for 48 hours, or (2) an indistinguishable placebo of 5% dextrose at identical intervals. Serial cultures were taken at operation and in the postoperative period. Antibiotic levels of fat were measured by tissue extraction and B. subtilis assay. After surgery, patients were monitored for infection by clinicians unaware of their random assignment group. The study was terminated and the code broken when it was evident that wound infections were increased to a statistically significant difference in one arm of the study. The brief perioperative administration of cefazolin reduced the incidence of wound infection from 21% to 4% (P less than 0.05) and the incidence of urinary tract and pulmonary infections from 17% to 0% (P less than 0.05). Tissue levels of antibiotics confirmed adequate dosages in the test subjects. The advantages of prophylactic administration of cefazolin for gastric bypass procedures are clearly demonstrated in this study. This confirms other reports which have shown prophylactic antibiotics to be useful in a variety of procedures. These studies suggest the need to reconsider the traditional opposition to prophylactic antibiotics and to determine whether perioperative antibiotics should be used routinely in all major operations.

Indigenous nasopharyngeal, auditory canal, and middle ear bacterial flora of gerbils: animal model for otitis media.

The indigenous microbial flora of the middle ear cavity of Mongolian gerbils, Meriones unguiculatus, was isolated, characterized, and identified, showing it to be sparse and transitory. Organisms, when found in the middle ear cavity, were most likely to be Staphylococcus epidermidis-like organisms. Cerumen from the external auditory canal of these animals yielded mostly staphylococci, coryneforms, and other gram-positive rods, including some anaerobic species. The nasopharynx supported a flora consisting mainly of staphylococci, lactobacilli, and coryneforms, with a smaller incidence and numbers of many other species. No mycoplasmas were cultured or seen in scanning electron microscope studies. None of the major pathogens of human otitis media were found; therefore, Mongolian gerbils are microbiologically acceptable candidates as a model for induced otitis media, using organisms isolated from human otitic infections.

Improved dispenser for use in preparing prereduced, anaerobically sterilized medium.

An improved, all glass, gravity-fed quantitative device for dispensing media during preparation of prereduced, anaerobically sterilized media with minimal loss due to oxidation of media is described.

Butyl rubber stoppers increase the shelf life of prereduced, anaerobically sterilized media.

Butyl rubber stoppers as compared with neoprene or black rubber stoppers significantly increased the shelf life of prereduced, anaerobically sterilized media for growth of obligately anaerobic bacteria.

Anaerobic bacteria in otitis media.

Anaerobic bacteria, Peptostrepotococcus intermedius and Propionibacterium acnes, were found in mixed culture specimens from four to ten tested cases of chronic secretory otitis media. These anaerobic bacteria were in a mixed infection flora with aerobic bacteria most often Staphylococcus epidermidis and Cornybacterium sp. which do not fit any established species. The findings of anaerobic bacteria in otitis media is consistent with the sporadic report of the involvement of anaerobic bacteria in otitis media in the literature since 1898.

Mobile anaerobe laboratory.

A laboratory cart equipped for obtaining clinical samples at bedside or operatory and for inoculating prereduced media for cultivation of medically important anaerobes is described.