Increasing the intra-Golgi pH of cultured LS174T goblet-differentiated cells mimics the decreased mucin sulfation and increased Thomsen-Friedenreich antigen (Gal beta1-3GalNac alpha-) expression seen in colon cancer.

Mucins in ulcerative colitis and colon cancer share common properties of reduced sulfation and increased oncofetal carbohydrate antigen expression. It has previously been shown that there is no simple correlation between these changes and the activity of the relevant glycosyl-, sialyl-, and sulfo-transferases. We examined mucin sulfation and expression of oncofetal Thomsen-Friedenreich (TF) antigen (galactosyl beta1-3N-acetylgalactosamine alpha-) in the goblet cell-differentiated human colon cancer cell line LS174T following treatment with bafilomycin A(1, )which raises intra-Golgi pH, or monensin, which disrupts medial-trans Golgi transport. Cells were dual-labeled with sodium [(35)S]-sulfate and D-[6-(3)H(N)]-glucosamine hydrochloride, or labeled with L-[U-(14)C]-threonine alone. Mucin was purified using Sepharose CL-4B gel filtration. Mucin sulfo-Lewis(a) and TF antigen expression were assessed using the F2 anti-sulfo-Lewis(a) monoclonal antibody and peanut agglutinin binding respectively. Bafilomycin (0.01 microM; 48 h) reduced total mucin sulfation, expressed relative to incorporation of glucosamine, to 0.50 +/- 0.04 d.p.m. [(35)S]-sulfate per d.p.m. [(3)H]-glucosamine compared to control, 0.84 +/- 0.05 (p < 0.001, n = 16). This was accompanied by 50.3 +/- 8.0% increased expression of TF antigen (p < 0.01) and 50.1 +/- 5.5% decreased expression of sulfo-Lewis(a) (p < 0.01). The reduced sulfate:glucosamine ratio was largely due to increased incorporation of glucosamine into newly synthesized mucin rather than reduction in total sulfate incorporation. In contrast, monensin only reduced total mucin glycosylation at concentrations > 0.1 microM and had no significant effect on mucin sulfation or TF expression. Intra-Golgi alkalinization affects mucin glycosylation, resulting in decreased mucin sulfation and increased expression of TF antigen, changes that mimic those seen in cancerous and premalignant human colonic epithelium.

A new kinetic model of protein adsorption on suspended anion-exchange resin particles.

The kinetics of adsorption of bovine serum albumin on an anion-exchange resin were measured in a batch system using a flow cell and ultraviolet absorbance, as a function of initial liquid-phase protein concentration and solid-to-liquid phase ratio. A new mathematical model for adsorption kinetics is presented that fits the experimental data to give a highly linear relationship with time, following a short transient period. Numerical integration of the differential form of the new composite nonlinear (CNL) kinetic model, containing three independent parameters, is shown to describe the dynamics of batch adsorption much better than alternative lumped parameter models. Although the new model is phenomenological rather than mechanistic, its principal parameter is shown to be a direct linear function of a physically measurable quantity. This study demonstrates that the model can accurately simulate protein concentration-time profiles using parameter estimates derived from correlations over a wide range of initial protein concentrations and phase ratios. The new CNL model is shown to be considerably superior to the Langmuir and solid-film linear kinetic models in this regard, having the additional advantage that an equilibrium isotherm for the system is not required.

Analysis of toxinogenic functions associated with the RTX repeat region and monoclonal antibody D12 epitope of Escherichia coli hemolysin.

Amino acids (aa) 550 through 850 of the Escherichia coli hemolysin (HlyA) contain sequences important for several steps in cytolysis. These include the Ca(2+)-binding glycine-rich tandem repeats recognized by the monoclonal antibody A10, the putative HlyC-dependent acylation site that corresponds to the monoclonal antibody D12 epitope, and the erythrocyte specificity domain which confers erythrolytic activity to the Pasteurella haemolytica leukotoxin. To further investigate the toxinogenic functions associated with this region of HlyA, we constructed mutants in the hlyA sequences coding for the repeat region and the D12 epitope. Mutants were analyzed for anti-HlyA antibody reactivity, cytolytic activities, target cell binding, Ca2+ requirements, and virulence. The D12 epitope was mapped to aa 673 through 726, with portions of the epitope both amino terminal and carboxy terminal to aa 700. This region was necessary, but not sufficient, for toxin binding to erythrocytes. A substitution at aa 684 resulted in loss of the D12 epitope, while cytolytic activity was retained. The nature of the D12 epitope and its associated functions are discussed. The A10 epitope mapped to residues 745 through 829, corresponding to repeats 4 through 11. Insertions within the glycine-rich repeats resulted in mutant forms of HlyA which retained A10 reactivity but required increased Ca2+ for lytic activity. These in vitro effects on cytolysis corresponded to a significant decrease in HlyA-mediated virulence in mice. HlyA from one insertion mutant was able to associate with leukocyte membranes under conditions that were Ca2+ deficient for cytolysis. The role of the glycine-rich repeats and Ca2+ in HlyA activity are discussed.

Assays of hemolytic toxins.

The ability to produce a cytolytic toxin contributes to the success of many organisms in a particular niche by such diverse means as lysis of a phagolysosomal membrane of the macrophage by hemolysin from the intracellular parasite Trypanosoma cruzi, disruption of leukocyte activity by the Escherichia coli hemolysin, and destruction of invading bacteria by hemolysin from the annelid Glycera dibranchiata. The relative contribution of erythrocyte lysis to survival of the cytolysin producer is still under investigation. Nevertheless, the hemolytic phenotype is both a powerful tool for identifying novel cytolysins and a convenient marker for studying cytolytic activity in established toxins.

Effects of gamma radiation and azathioprine on Brucella abortus infection in BALB/c mice.

Sublethal irradiation of BALB/c mice 4 hours prior to inoculation with 5 x 10(4) virulent Brucella abortus, caused significant (P less than 0.01) reductions in bacterial numbers in comparison with numbers in unirradiated controls. Numbers of brucellae in the spleen were significantly lower by 5 days after inoculation and decreased thereafter, so that at 2 and 3 weeks after inoculation, there were up to 1,000-fold fewer organisms in the spleen of irradiated mice. The number of brucellae in the spleen increased in irradiated mice thereafter. The course of events in the liver was similar, but developed more slowly, and peak differences in bacterial numbers were about 1 log less. These phenomena were not attributable to differences in implantation of brucellae in the liver or spleen, nor to an abnormal distribution of organisms in other organs of irradiated mice. Irradiation of mice during the plateau phase of infection also resulted in significant (P less than 0.05) reductions in bacterial counts in the spleen during the succeeding 4 weeks. Macrophage activation in the spleen, measured by a Listeria monocytogenes-killing assay, was significantly (P less than 0.01) increased by irradiation alone at 1 week after inoculation and at that time was significantly (P less than 0.01) greater in B abortus-infected, irradiated mice than in B abortus-infected controls. Histologic, cytologic, and immunologic studies revealed that the decrease in numbers of organisms between 1 and 2 weeks after inoculation in irradiated mice occurred at a time when their immune response to B abortus was suppressed and when numbers of neutrophils and monocytes infiltrating the spleen were significantly (P less than 0.01) diminished.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative histopathology in BALB/c mice infected with virulent and attenuated strains of Brucella abortus.

The course of infection in BALB/c mice of virulent Brucella abortus strain 2308 (S-2308) and attenuated strain 19 (S-19) varies markedly. Whereas S-19 is eliminated at an exponential rate beginning at 2 weeks post infection (p.i.), strain 2308 assumes a steady state or plateau during the first 6 weeks p.i. and thereafter is eliminated very slowly over a period exceeding 6 months. Here we compared the initiation and maintenance of inflammatory reactions in spleens and livers of mice infected with either of the two strains of B. abortus for the first 6 weeks p.i. Histological changes in the liver were similar in response to either strain and were characterized by the development of small granulomas and an influx of polymorphonuclear leukocytes (PMN) and monocytes. Tissue reactions in the spleen were similar at weeks 1 and 2 p.i. At 3 weeks p.i. and thereafter, focal granulomatous responses in S-2308-infected mice exceeded those in mice infected with S-19. Numbers of nonspecific esterase (NSE) positive mononuclear leukocytes in S-19-infected spleens had increased by 3 weeks p.i. and remained elevated. No comparable increase in NSE positive cells occurred in mice infected with S-2308, and numbers were significantly lower. At 4 weeks p.i. the influx of mature neutrophils and the intensity of extramedullary hematopoiesis were significantly greater in S-19-infected spleens. A profound depletion of periarteriolar lymphoid tissue was noted in both infections for the first 3 weeks p.i. However, repopulation of lymphoid sheaths in S-19-infected spleens became significantly greater by 4 weeks p.i. and continued to increase at significantly higher levels during the next 2 weeks. This study demonstrates quantitative differences in splenic inflammatory responses which are temporally related to the more rapid elimination of S-19. Based upon the lower susceptibility of strain 2308 to the protective effects of immune serum it is hypothesized that the different patterns of infection and inflammation displayed by the 2 strains may related to the differential capacities of antibody opsonized S-19 and S-2308 to survive in activated macrophages.

Temporal development of protective cell-mediated and humoral immunity in BALB/c mice infected with Brucella abortus.

In BALB/c mice infected i.v. with attenuated strain 19 of Brucella abortus, the organism replicates to high numbers in the spleen and reaches peak concentrations at 2 wk postinfection (p.i.). The infection is then progressively cleared so that by 8 wk p.i. numbers of bacteria have decreased 10,000 fold or more. Passive transfer assays were performed with T cell-enriched spleen cells and serum of donor mice infected 2, 3, 4, 5, 6, or 8 wk previously. Antibodies conferred significant protection to recipients at and after 3 wk p.i., whereas protection by T cells was not evident until 4 wk p.i. The combined transfer of serum and cells enhanced protection over that provided by serum or cells alone when transfers were made before, but not after, challenge infection. Protection conferred by T cell-enriched spleen cells of 6-wk donors was unaffected by the presence of equal quantities of cells from 3-wk donors, but was abrogated by the removal of both CD4 and CD8 T cell subsets. Experiments with purified CD4 and CD8 subsets revealed that cell-mediated protection resided at equivalent levels in both subsets. Daily treatment of mice with Cyclosporin A for 4 wk after infection caused some increase in numbers of brucellae in spleens and livers. Although immune responses of treated animals were markedly suppressed, there was little effect of treatment on numbers of macrophages in the spleen, on enhanced killing of Listeria monocytogenes in the spleen, or on the nature and intensity of splenic and hepatic inflammatory responses. These data indicate that acquired resistance to infection with B. abortus in mice is the result of independent, and probably also interactive, effects of antibodies and T effector cells of both CD4 and CD8 phenotypes. The initial decline in bacterial numbers in the spleen, which occurred in the absence of detectable cell-mediated immunity in that organ, could probably be ascribed principally to effects of antibodies and to nonimmune stimuli responsible for increased formation, attraction, and activation of macrophages.

Effectiveness of natural and synthetic complexes of porin and O polysaccharide as vaccines against Brucella abortus in mice.

A single vaccination of mice with a complex of porin and smooth lipopolysaccharide (porin-S-LPS) extracted from virulent Brucella abortus 2308 provided significant protection (P less than 0.01 to P less than 0.001) against challenge with the same strain, equivalent to that achieved by vaccination with living attenuated B. abortus 19. The porin-S-LPS vaccine given without adjuvant or in several adjuvants (trehalose dimycolate and muramyl dipeptide; the pluronic polymer L-121 and muramyl dipeptide; or complexed with Quil A in immunostimulating complexes) provided equivalent protection. In contrast, one vaccination with porin complexed with rough LPS (porin-R-LPS) from a rough mutant of strain 2308 provided no protection with any adjuvant tested. In one experiment, two inoculations with the porin-R-LPS resulted in a low level of protection, probably owing to priming of the animals for production of O-polysaccharide-specific antibodies. However, one vaccination with rough-strain porin covalently bound to purified O polysaccharide conferred protection equal to that obtained with natural complexes of porin-S-LPS or with living strain 19. A synthetic vaccine containing long chains of O polysaccharide was more effective than one prepared with short chains. Protective vaccines caused the formation of increased concentrations of circulating O-polysaccharide-specific antibodies, although there were individual exceptions to the quantitative association between O-polysaccharide-specific antibodies and protection. Antibodies specific for porin or R-LPS were found in negligible quantities in vaccinated mice. These results provide additional evidence that the O polysaccharide will constitute an essential component of an effective subcellular vaccine against B. abortus and that O-polysaccharide-specific antibodies play an important role in protective immunity in brucellosis.

Comparative immune responses to native cell envelope antigens and the hot sodium dodecyl sulfate insoluble fraction (PG) of Brucella abortus in cattle and mice.

Brucella abortus vaccines composed of native cell envelopes or outer membrane proteins of smooth strain 2308 were compared with a vaccine (PG) composed of the insoluble residue of strain 2308 cell envelopes which had been extracted with hot sodium dodecyl sulfate. Vaccines were given by injection in an oil base adjuvant containing trehalose dimycolate and muramyl dipeptide or without adjuvant. Mice vaccinated with 30 micrograms native cell envelopes or PG and challenged 4 weeks later with virulent B. abortus strain 2308 displayed equivalent levels of protective immunity at 1 and 4 weeks post-infection. Heifers were vaccinated with 5 mg of antigens in adjuvant; PG was also administered without adjuvant. Humoral and cell mediated immune (CMI) responses were tested at monthly intervals. PG without adjuvant induced negligible immune responses. Native cell envelope antigens induced significantly higher titers of whole cell agglutinins over a 3-month period than did PG, although revaccination with PG in adjuvant enhanced the production of agglutinins and both vaccines induced antibodies to the O polysaccharide. Lymphocyte blastogenesis responses and delayed hypersensitivity reactions to porin and group 3 proteins were stimulated by both native and PG vaccines, and the magnitude of the responses did not differ significantly between the treatment groups. These vaccines were therefore comparable in their capacity to induce protective immunity in mice and CMI responses in cattle, whereas antibody responses induced by PG in cattle were generally lower. These findings provide a basis for evaluation of nonliving B. abortus vaccines in cattle.

Antifungal properties of 2-n-alkyn-1-ols.

Fourteen 2-n-alkynols (C3-C14, C16, and C18) were tested against Aspergillus oryzae, Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 5.6 and 7.0. Toxicity to Candida albicans, Candida tropicalis, Trichophyton mentagrophytes, and Mucor mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. Fungitoxicity was strongly influenced by chain length, slightly by the pH of the medium, and significantly by the presence of beef serum. 2-n-Undecyn-1-ol was the most active member of the series, and there was marked synergism between it and ketoconazole.