A whole-organ perfusion model of Bordetella pertussis adherence to mouse tracheal epithelium.

A whole-organ perfusion system was used to culture tracheas from adult Swiss mice and test this system's adaptability for use in adherence assays for virulent Bordetella pertussis. Culture medium and bacterial suspensions flowed readily through the tracheal lumen, ciliary activity was maintained throughout the culture period, and scanning electron microscopy revealed retention of normal surface morphology. The number of adherent colony-forming units (cfu) per trachea was determined for all three Bordetella species every 30 min over a 3.5-h incubation period and the resultant adherence patterns were reproducible. Adherent cfu were dependent on the concentration of microorganisms in the infecting inoculum. Bordetella pertussis did not demonstrate a preferential adherence to either the dorsal or ventral surface of the tracheal epithelium nor did it demonstrate a preference for adherence to the laryngeal or bronchial end of the trachea. Static growth conditions did alter the adherence pattern of B. pertussis from that observed when the organism was grown with constant agitation.

Scanning electron microscopy of mouse ciliated oviduct and tracheal epithelium infected in vitro with Bordetella pertussis.

Infection of mouse tracheal organ culture with Bordetella pertussis resulted in ciliostasis within 36 h. Scanning electron microscopy revealed that B. pertussis attached exclusively to ciliated cells but did not induce expulsion of this cell type at a test interval of 48 h. Mouse oviduct organ culture infected with B. pertussis demonstrated the same strict tropism for ciliated cells as in the tracheal ring system. Only ciliated cells were parasitized, becoming heavily colonized 48 h postinfection. Infected ciliated oviduct cells were not extruded. A fixation method which enhances fine structure was used in the scanning electron microscope studies. Bacterial fimbriae were not observed as the method of attachment of B. pertussis to cilia but fine fibers were seen extending between cilia and bacterial cells.

Gas-Liquid Chromatographic Differentiation Between Salmonella gallinarum and Salmonella pullorum.

Salmonella gallinarom and Salmonella pullorom have been considered as one serovar, S. gallinarom-pullorom or S. gallinarom . This serovar possesses group D somatic antigens with no flagellar antigen. Reportedly S. gallinarom differs from S. pullorom in dulcitol fermentation. This reaction is positive, but delayed up to 5 d for S. gallinarom and negative for S. pullorom . Gas-liquid chromatography of organic acid by-products from a dulcitol medium was performed on 10 isolates of each biovar. Viable plate counts confirmed approximately the same number of organisms per ml of culture. Results of pH determinations supported gas-liquid chromatographic analysis of more acid formation in all S. gallinarom cultures as compared with the S. pullorom cultures after incubation for 24 h. A quantitative measurement of succinic acid resulted in confirmation of the differences in metabolic function of both biovars. The additional test procedure of gas-liquid chromatography of organic acid by-products aids the clinician or researcher in rapidly and accurately distinguishing these two similar biovars.

Chemical carcinogenesis and immunity: immunologic status of rats treated with methylnitrosourea.

During the treatment of outbred Sprague-Dawley rats with methylnitrosourea (MNU) or the noncarcinogenic analog diphenylnitrosamine, antibody levels to teichoic acid as well as several parameters of lymphocyte and macrophage function were assessed in animals not overtly stimulated with antigen. Treatment with MNU did not appear to alter most immunologic parameters studied. Some alterations occurred in natural antibody levels, in spleen weight, and in peripheral blood differentials of rats that had received the highest carcinogen dose (4.5 mg/kg). These alterations, however, appeared to coincide with the development of tumors and were not observed in animals with premalignant lesions. Levels of chemical carcinogens that were capable of inducing tumors did not necessarily suppress the immune response.

Direct identification of the murine pluripotential stem cell using rabbit anti-mouse brain serum.

Previous studies have shown that antisera prepared in rabbits against mouse brain tissue (RAMBS) contain activity against the murine bone marrow colony-forming unit (CFU-s) or pluripotential hemotopoietic stem cell. In the present study, the F(ab')2 portion of RAMBS was examined for its potential efficacy in the identification of the mouse CFU-s when used in an indirect immunofluorescence-labeling technique. After separation of mouse bone marrow cells by a discontinuous bovine serum albumin density gradient, fluorescent cells were observed only in those bands which, by the splenic colony-forming assay, demonstrated CFU-s. Furthermore, the quantity of CFU-s demonstrated by the spleen colony-forming assay approximated the number of fluorescent cells observed in the corresponding band. It appears that the F(ab')2 portion of RAMBS used in an immunofluorescent assay may provide a method for the direct quantitation and identification of the CFU-s content of murine bone marrow.

Effect of iodoacetate on T lymphocytes of young AKR mice.

The influence of sulphydryl inhibitor iodoacetate on properties characteristically associated with T-lymphocytes was examined in young AKR mice. Thymocytes from mice receiving 100 microgram of iodoacetate showed a decrease in cortisone sensitivity, and responded more vigorously to phytohemagglutinin (PHA). Spleen cells from treated mice also exhibited greater reactivity to both PHA and concanavalin A (Con A). The uptake of 14C-iodoacetate by the thymus, spleen, lymph nodes, bone marrow, liver and kidneys also was investigated. One hour, 2 h and 18 h after the intraperiotoneal injection of 10-3 M iodoacetate, the thymus demonstrated a higher incorporation of the labelled compound than did the spleen, lymph nodes or other organs examined. These findings provide further evidence to indicate that the immuno-enhancement observed following iodoacetate treatment is related to its effect on T lymphocytes.

Iodoacetate-induced inhibition and enhancement of spontaneous leukemia in AKR mice.

Either inhibition or enhancement of the spontaneous lymphoma exhibited by AKR mice was observed after treatment with different dosages of the sulfhydryl inhibitor, sodium iodoacetate. Treatment of the mice at 3 or 6 months of age with five ip injections of 0.10 mg of iodoacetate at 5-day intervals significnatly extended the survival of the animals. A single administration of this dosage elevated the responses of splenic lymphocytes to the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A), and resulted in higher PHA and Con A response ratios than were noted for age-matched controls. Conversely, groups of 3- or 6-month-old AKR mice, subjected to the same regimen but with 0.01-mg dosages, exhibited an apparent accelerated development of the leukemia and survived for significantly shorter periods. In general, splenic lymphocytes harvested from mice given a single 0.01-mg treatment of iodoacetate were not as reactive as were control cell cultures when exposed to the T-cell mitogens.

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.

Antigenic cross-reactivity among rodent brain tissues and stem cells.

Previous studies have shown that antisera prepared in rabbits against mouse brain (RAMB) contains activity in vitro against the mouse bone marrow colony-forming unit (CFU) or hematopoietic stem cell. In the present study, in vitro treatment of mouse bone marrow with antisera prepared in rabbits against brain tissue from rats (BARB) or hamsters (RAHB) also reduced the CFU content of the mouse marrow. Prior absorption of the RAMB serum with fetal liver tissues from rats or hamsters as well as mice reduced the anti-CFU activity of the RAMB preparation. In addition, absorption of the RAMB preparation with brain tissue from any of the rodents reduced the activity of the antiserum for the mouse stem cell. It appears that the determinants previously shown to be shared by the brain tissue and stem cells of mice are cross-reactive with determinants present on the stem cells and brain tissue of rats and hamsters.

Identification of hamster thymus-derived lymphocytes employing rabbit anti-hamster brain serum.

Rabbit anti-hamster brain sera prepared from the brains of Syrian and Chinese hamsters were evaluated for their potential to identify thymus-derived lymphocytes present in these animals. The unabsorbed antisera were cytotoxic in vitro for both homologous and heterologous thymocytes and splenocytes. Following absorption with hamster liver and erythrocytes, the antisera remained toxic for thymocytes, but were less toxic for splenocytes. Comparative testing of both the rabbit anti-Syrian and anti-Chinese brain sera with rabbit anti-C3H mouse brain serum indicated that considerable cross-reactivity exists between the different anti-brain sera. The in vivo administration of any of the antisera to C3H mice resulted in a lowered titer to the thymus-dependent sheep erythrocyte antigen response as measured by the hemolysis-in-gel technique. In vitro treatment with the anti-brain sera had no effect on plaque-forming cells present in the spleen.

Effect of iodoacetate on the bone marrow immunocompetence of AKR mice.

Studies were conducted to determine whether the sulfhydryl inhibitor, sodium iodoacetate, administered to preleukemic AKR mice and to mature C3H mice altered the immunocompetence of their bone marrow. Parameters investigated included the splenic plaque-forming capacity directed to sheep erythrocytes of bone marrow transferred from iodoacetate-treated aanimals to irradiated syngeneic recipients and the mitogenic responsiveness of bone marrow cells from untreated and iodoacetate-treated preleukemic AKR mice to phytohemagglutinin and concanavalin A. The administration of two 0.5-ml doses of 10 mM iodoacetate to preleukemic AKR mice and to C3H mice resulted in a significant increase in bone marrow immunocompetence. Irradiated mice given marrow transplants from iodoacetate-treated syngeneic donors exhibited greater numbers of plaque-forming cells directed against sheep erythrocytes than did recipients of marrow from control animals. This effect was abrogated when the donor marrow was previously treated in vitro with rabbit antimouse brain serum and the complement to remove thymus-derived lymphocytes. The mitogenic responsiveness of marrow cultures from iodoacetate-treated AKR mice to phytohemagglutinin was similar to that observed for control mice, while the response to concanavalin A was decreased. These findings suggest that the administration of iodoacetate potentiated the immunocompetence of bone marrow by affecting thymus-derived cells.

In vivo effects of rabbit anti-mouse brain serum on theta bearing lymphocytes of AKR mice.

The in vivo effect of rabbit anti-AKR mouse brain-associated serum (RAMB) was determined on theta bearing lymphocytes present in the spleens and thymuses of mature C3H mice and AKR mice staged into preleukemic, leukaemic and overtly leukaemic states. Following seven daily injections of RAMB serum, the splenic plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) and the percentage of theta-bearing lymphocytes in the spleen were significantly decreased in the C3H and the preleukaemic AKR mice. Decreases in thymic weight and thymocyte numbers were also apparent. Determinations of theta antigen density using in vitro cytoxicity tests indicated that splenic and thymic T lymphocytes (thymus-derived) remaining in the RAMB-treated C3H and preleukemic AKR mice consisted primarily of cells bearing less of the theta surface antigen. Histopathological studies of tissues from these two treated groups revealed cortical lymphocyte depletion in the thymus, and marginal and periarteriolar depletion in the spleen. Leukaemic AKR mice, administered seven injections of RAMB serum, demonstrated less dramatic changes in thymus weight, histopathology and theta-bearing cell percentages when compared with the data from the preleukaemic AKR or matureC3H mice. The results from testing overtly luekaemic AKR mice administered RAMB serum for the 7 or 9 days did not demonstrate differences from findings from groupsof overtly leukaemic control mice. These data indicate that the in vivo activity of RAMB serum in C3H mice and preleukaemic AKR mice is directed primarily toward the less mature T-lymphocyte population. This influence of RAMB serum is lesspronounced in leukaemic and the more overtly leukaemic mice, suggesting that a decreasedpopulation of RAMB-susceptible lymphocytes are present in these animals.