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[Effect of prodigiozan and lysozyme on the surface structures of macrophages after immunosuppression].

The state of macrophage surface structures is closely connected with the function 1 capacity of these cells. Peritoneal macrophages from mice subjected to strong 10-day immunosuppression with azathioprine (60 mg/kg), prednisolone (50 mg/kg) or their combination (25 and 30 mg/kg, respectively) were investigated with scanning electron microscopy. Azathioprine and especially its combination with prednisolone induced pronounced degenerative affections in the macrophages and lymphocytes, smoothing of their surfaces, predominance of large spread macrophages, and impairment of the intracellular cooperation. The macrophage alteration under the action of prednisolone was less pronounced. Prodigiozan, a bacterial polysaccharide (0.5 mg/kg) administered on the 4th and 9th days of the immunosuppression increased the macrophage resistance mainly to the damaging effect of prednisolone and was low effective after administration of azathioprine. The effect of lysozyme injected intramuscularly in a dose of 0.5 mg/kg daily within a period of the 3rd to the 10th days of the use of the immunosuppressants was evident from activation of the surface structures of the small macrophages and lymphocytes and reduction of cell alterations independent of the type of the immunosuppressants used. This indicates the necessity of a differential approach to the use of the macrophage activators for correcting the decreased infection resistance after medicamentous immunosuppression.

Animals↗

[Cytochemical study of peroxidase activity and the peroxidase-endogenous hydrogen peroxide system in the peripheral blood neutrophils of mice exposed to prodigiozan and retabolil].

The paper concerns changes in the activity of some enzymes of azurophil granules of mouse neutrophils under a single administration of the bacterial polysaccharide prodigiosan and the synthetic anabolic hormone retabolil. Prodigiosan increases 1.4-fold the activity of the peroxidase-hydrogen peroxide system in azurophil granules of intact mice. In mice with leukopenia, prodigiosan normalizes the leukocyte count after 24 hours. At the same time there is an increase in the activity of peroxidase. It is assumed that under the effect of the same drug the cells of the animals in different physiological conditions may show an activation of the different components of antimicrobial defence. Retabolil raises the activity of peroxidase 96 hours after the first administration. Following 192 hours the activity reduces to normal and remains unchanged after the second drug administration.

Anabolic Agents↗

[Avian yolk antibodies in diagnosis and research].

Hens were immunized with bacterial polysaccharide (alginate), Hepatitis B surface antigen (HBsAg), and potato viruses (PVA, PVS, PVM, PVX, and PVY). The antibodies were isolated noninvasively from the yolks of laid eggs. The purified yolk immunoglobulins (IgY) were tested in an array of various assays and diagnostic techniques. The methods employed were precipitation reactions, immun-electrophoresis, ELISA (after biotinylation of IgY), immuno-gold electron microscopy, and western and immuno blotting. Some of these methods had to be modified according to the special requirements of avian antibodies. The special handling of this animal system is described in regard to antibody production. The results demonstrate that IgY derived from hens can replace IgG produced by traditional methods in mammals. The advantages of this alternate animal system are emphasized in respect to animal care, high productivity, and special suitability of avian antibodies for certain diagnostic purposes.

Animals↗

Phase I study of the antineovascularization drug CM101.

CM101 is a bacterial polysaccharide that induces neovascular inflammation in malignant tumors. Fifteen patients with refractory malignancies received CM101 i.v. by a 15-min infusion every other day, three times in 1 week, at doses ranging from 1 unit (7.5 microgram)/kg to 5 units/kg. Serum was analyzed for anti-CM101 IgG and IgM weekly. Plasma levels of inflammatory cytokines, including tumor necrosis factor alpha, interleukin 8, interleukin 10, MIP-1alpha, and soluble E-selectin, were analyzed from -15 min to 12 h during each treatment. Dose-limiting toxicities, including grade IV dyspnea and arrhythmia, were encountered at the 5-unit/kg level. Toxicities occurred primarily within the first 12 h after therapy and included mild-to-moderate fever and chills, nausea, cough, headache, facial flushing, dyspnea, myalgias, and acute tumor-related pain. No patient developed detectable antibodies to CM101. All patients experienced marked time- and dose-dependent elevations in all cytokines studied. Three patients experienced tumor shrinkage. The results show that CM101 can be safely administered at doses that produce evidence for severe, and possibly tumor-specific, inflammation. Further study is necessary to better characterize the mechanism of action and determine the optimal dose and schedule of this new agent.

Adult↗

An alternative infrared spectroscopy assay for the quantification of polysaccharides in bacterial samples.

The ability of bacteria to produce extracellular polysaccharides has been regarded as an indication of biofilm-forming capacity. Therefore, the determination of the sugar content in bacterial samples becomes a significant parameter. The colorimetric methods currently used are rather sensitive to the nature of the sugars and therefore require knowledge of the sugar types present in the samples. Unfortunately, the types of sugars present in bacteria are generally unknown and often composed of a complex mixture. In this article, we propose an alternative method based on Fourier transform infrared (FTIR) spectroscopy for the estimation of the total sugar content in bacterial samples. The method is based on a systematic treatment of FTIR spectra obtained from dried bacteria samples. It is assumed that the total sugar amount can be estimated from the area of characteristic bands between 970 and 1182 cm(-1). In parallel, the amide II band (1560-1530 cm(-1)) associated with proteins, or the C-H stretching region (2820-3020 cm(-1)) associated with the biomass, can be used for normalization purposes. Therefore, the ratio of the band area in the sugar window over that of the amide II or C-H stretching can be used to report the sugar content in bacterial samples. This method has been validated on model bacterial mixtures containing sugars, proteins, and DNA. Results with real bacterial samples are also provided and show conclusively that increased sugar contents in biofilms can be identified. The proposed FTIR approach requires minimal sample preparation and a single acquisition, is rapid, and may be applied to any kind of bacterial growth.

Animals↗

Interactions between Brucella melitensis and human phagocytes: bacterial surface O-Polysaccharide inhibits phagocytosis, bacterial killing, and subsequent host cell apoptosis.

Brucellae are gram-negative intracellular pathogens that survive and multiply within host phagocytic cells. Smooth organisms present O-polysaccharides (OPS) on their surface. The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth, virulent B. melitensis 16M results in a rough mutant designated WRR51. Unlike B. abortus, both smooth and rough strains of B. melitensis are resistant to complement-mediated killing. To determine the role of surface OPS in the interactions of B. melitensis with monocytes/macrophages (M/M), 16M and WRR51 were transformed with the plasmid pBBR1MCS-6y encoding green fluorescent protein, and the transformants were used to infect human mononuclear phagocytes with and without fresh human serum as a source of complement. Human monocytes were cultured in the presence of macrophage colony-stimulating factor to allow their differentiation into macrophages during the course of infection. Intracellular bacteria were easily visualized using fluorescence microscopy. Infection in M/M, identified by surface staining and fate of infected phagocytes, was quantitated by flow cytometry. Rough bacteria were internalized, with no requirement for opsonization by serum, at a higher rate than smooth organisms. Smooth B. melitensis survived and multiplied for at least 6 days inside M/M, but rough organisms were eliminated by death of the infected cells. In human monocytes cultured for 1 day without serum in order to trigger the apoptotic pathway, infection by rough brucellae accelerated phagocyte death; smooth brucellae inhibited apoptosis. This study suggests that the presence of surface OPS on live B. melitensis benefits the bacterium by preventing the death of macrophages, Brucella's preferred target for intracellular replication.

Apoptosis↗

Synthesis of beta-(1-->6)-linked glucosamine oligosaccharides corresponding to fragments of the bacterial surface polysaccharide poly-N-acetylglucosamine.

A series of 3-beta-acetamidopropyl oligo-beta-(1-->6)-glucosamines consisting of 5, 7, 9 and 11 glucosamine residues, and a series of corresponding per-N-acetylated derivatives were synthesized using a convergent blockwise approach. These compounds represent fragments of a bacterial surface polysaccharide produced by numerous bacterial pathogens, including Staphylococcus aureus, and will be used as models for its biochemical and immunological properties.

Acetylglucosamine↗

Modulation of surgical fibrosis by microbial zwitterionic polysaccharides.

Bacterial carbohydrates have long been considered T cell-independent antigens that primarily induce humoral immune responses. Recently, it has been demonstrated that bacterial capsules that possess a zwitterionic charge motif can activate CD4(+) T cells after processing and presentation by antigen-presenting cells. Here we show that these zwitterionic polysaccharides can prevent T helper 1-mediated fibrosis by signaling for the release of IL-10 from CD4(+) T cells in vivo. IL-10 production by these T cells and their ability to prevent fibrosis is controlled by the inducible costimulator (ICOS)-ICOS ligand pathway. These data demonstrate that the interaction of the zwitterionic polysaccharides with T cells results in modulation of surgical fibrosis in vivo and suggest a previously undescribed approach to "harnessing" T cell function to prevent inflammatory tissue disorders in humans.

Animals↗

The affinity of bacterial polysaccharide-containing fractions for mammalian cell membranes and its relationship to immunopotentiating activity.

The natural affinity of various bacterial glycopeptides and lipopolysaccharides for mammalian cell membranes was estimated quantitatively by comparison with the adsorption of lipopolysaccharide from Escherichia coli NCTC 8623 to erythrocytes, thymocytes, bone marrow cells, spleen cells, peritoneal lymphocytes and macrophages. Immunopotentiating activity was estimated by measuring the ability of the bacterial fractions to stimulate a humoral response to ovalbumin in HAM/1CR mice. When the affinity for mammalian cell membranes was compared with the stimulation of the antibody response, it was found that a negative correlation for peritoneal macrophages (rs = -0.94, P less than 0.0005) and a positive correlation for peritoneal lymphocytes (rs = +0.97, P less than 0.0005) and spleen cells (rs = +0.76, P less than 0.005) existed.

Adjuvants, Immunologic↗

Heterogeneity in bacterial surface polysaccharides, probed on a single-molecule basis.

The heterogeneity in bacterial surface macromolecules was probed by examining individual macromolecules on the surface of Pseudomonas putida KT2442 via single-molecule force spectroscopy (SMFS). Using an atomic force microscope (AFM), the silicon nitride tip was brought into contact with biopolymer molecules on bacterial cells and these macromolecules were stretched. Force-extension measurements on different bacterial cells showed a range of adhesion affinities and polymer lengths. However, substantial heterogeneity was also observed in the force-extension curves on a single bacterium. A given bacterium has biopolymers that range in size from tens to hundreds of nanometers, with adhesion affinities for the AFM tip from nearly zero to greater than 1 nN. A distribution of polymer sizes was confirmed by size-exclusion chromatography. The freely jointed chain (FJC) model for polymer elasticity was applied to individual force-extension curves in order to estimate the contour lengths and segment lengths of the polymer chains. A range of segment lengths was obtained using the FJC model, from 0.154-0.45 nm in water, 0.154-0.32 nm in 0.01 M KCl, and 0.154-0.65 nm in 0.1 M KCl. The modeling confirms that the heterogeneity in biopolymers is more than a matter of differences in molecular weights, since a range of stiffnesses (segment lengths) was also observed. The effect of salt concentration on biopolymer conformation and adhesion was also explored. While the biopolymers were flexible in all solvents, they were slightly more extended in water than in either of the salt solutions (0.01 and 0.1 M KCl). The adhesion of polysaccharides with the AFM tip was not dependent on salt concentration, because the polymers were not highly charged and heterogeneity overwhelmed any trends that could be observed in adhesion with respect to solution ionic strength. These experiments indicate that heterogeneity in biopolymer properties on an individual bacterium and within a population of bacterial cells may be much greater than previously believed and should be incorporated into models of bacterial adhesion.

Bacterial Adhesion↗

Thermolytical techniques to characterize fungal polysaccharides and bacterial lipopolysaccharides.

The present work constitutes an entirely novel contribution in the scope of microbiology and especially in taxonomy, introducing thermolysis curves as a rapid method of characterization of fungal polysaccharides and bacterial lipopolysaccharides. The thermal analysis techniques applied were thermogravimetry and derivative thermogravimetry (TG-DTG), differential thermal analysis (DTA), and differential scanning calorimetry (DSC). Each thermogram of a sample is represented by one or a few temperatures and, in DSC, by complementary enthalpy data. The temperatures of the thermograms from structurally unknown polysaccharides are compared with those used as references, and thus, information on their composition, linkage types, and anomeric configuration can be deduced. The situation is more complicated for bacterial lipopolysaccharides, but in whatever mode, a structural estimation is always possible. In the course of the development and validation of the thermal method, structural findings on relative stabilities of linkage types (valuable in carbohydrate research) have been recognized and are therefore also described in this work.

Biotechnology↗

A new method of non-cross-linking conjugation of polysaccharides to proteins via thioether bonds for the preparation of saccharide-protein conjugate vaccines.

Bacterial polysaccharides, including capsular polysaccharides, are poor immunogens particularly in young infants. However, conjugation of bacterial polysaccharides to immunogenic carrier proteins generally results in conjugates that induce strong antipolysaccharide T-helper-cell dependent immune responses, also in young infants. The magnitude of the response and the extent of the T-helper-cell dependency is related to the chemical characteristics of the particular conjugate such as presence or absence of polysaccharide-protein cross-linking, presence or absence of spacer arms, character of spacer arms, type of carrier protein, size of conjugated polysaccharide hapten and molar degree of substitution. In the present study a new, general and simple method for the preparation of poly- and oligosaccharide-protein conjugates is presented. This new method is based on spacer-introducing chemistry that allowed for conjugation of a model polysaccharide, dextran, ranging in size from 0.5 to 150 kDa, to tetanus toxoid (TTd). The developed conjugation method involves derivatization of polysaccharide with 2-iminothiolane (2-IT) and activation of carrier protein, such as TTd, with N-hydroxysuccinimide ester of bromoacetic acid. Reaction rates and accordingly the substitution of the conjugates, could be controlled by varying time, pH and concentration of the reactants. Unlike direct reductive amination, the 2-IT based conjugation technology is fast and made it possible to couple fairly large polysaccharides to TTd.

Dextrans↗

Evolution of the subclass of IgG antibody to type 3 pneumococcal polysaccharide during childhood.

Human antibodies to bacterial polysaccharides consist primarily of IgG and are largely restricted to the IgG2 subclass in adults. We examined the ontogeny of the IgG subclass response to pneumococcal polysaccharide type 3 to determine if the poor response of infants to immunization with polysaccharide antigens is due to a diminished capacity to form this subclass of antibodies. Sera from 33 patients aged 2 months to 25 years who had previously been shown to respond to polyvalent pneumococcal polysaccharide vaccine by producing IgG antibodies, were assayed for pneumococcal type 3 specific antibodies of the IgG1, IgG2, IgG3, or IgG4 subclass. IgG1 antibodies to pneumococcal polysaccharide type 3 were uniformly low in all age groups. In contrast, IgG2 antibody activity was lowest in children less than the age of 2 years (170 +/- 20 ng/ml), but rose progressively in the age group 2-5 years (210 +/- 40 ng/ml), 5-10 years (330 +/- 30 ng/ml), and over the age of 10 (390 +/- 30 ng/ml) (differences significant at P less than 0.0005 by ANOVA). Thus, even in infants, pneumococcal polysaccharide responses are confined largely to the IgG2 subclass. Our findings are consistent with the hypothesis that purified bacterial capsular polysaccharide antigens preferentially activate IgG2-committed B cell clones at all ages.

Adolescent↗

Antibodies to Haemophilus influenzae type b polysaccharide affect bacterial adherence and multiplication.

Since immunization of infants with conjugated Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) vaccines results in a reduction of colonization, we determined the inhibitory effect of anti-Hib PS on two steps in the colonization, i.e., adherence of H. influenzae to nasopharyngeal epithelium and bacterial growth. Monoclonal antibody (MAb) E117-5 specific for Hib PS inhibited at a concentration of at least 80 microg/ml in vitro the adherence of Hib strain 770235f+b+ to oropharyngeal epithelial cells by 50% (P <, 0.02), but this MAb and sera from children immunized with Hib PS conjugate vaccine (n = 10) were not inhibitory in final dilutions containing up to 20 microg of anti-Hib PS per ml. The growth of Hib strain 770235f+b+ did completely stop in the presence of 5 microg of anti-Hib PS MAb E117-5 per ml and human sera with an anti-Hib PS concentration of 2 microg/ml or more, in contrast to the growth of the nonencapsular variant strain 770235f+b0.

Agglutination↗

Comparison of active and combined passive/active immunization of Navajo children against Haemophilus influenzae type b.

In a high risk Navajo population we compared the immunogenicity of a new Haemophilus influenzae type b mutant-diphtheria toxic conjugate vaccine (HbOC) with simultaneous active (HbOC) and passive immunization with bacterial polysaccharide immunoglobulin prepared from adults immunized with H. influenzae b, pneumococcal and meningococcal vaccines. Only 7 of 26 (27%) 2-month-olds had an increase in H. influenzae b capsular polysaccharide antibody after a single dose of HbOC, a proportion similar to that of saline controls (9 of 25, 36%). After a second HbOC dose at 4 months 88% had antibody concentrations of 0.15 microgram or more, and after a third dose at 6 months all had antibody levels greater than or equal to 0.15 microgram/ml. The group receiving both HbOC and bacterial polysaccharide immunoglobulin at 2 months uniformly had H. influenzae b CP antibody concentrations of greater than or equal to 0.15 microgram/ml at 4 months (P less than 0.001 relative to "HbOC alone" group) and subsequently responded similarly to second and third doses of HbOC vaccine as did also the "HbOC alone" group. We conclude that combined passive/active immunization with bacterial polysaccharide immunoglobulin and HbOC at 2 months maintains antibody at concentrations thought to be protective (greater than or equal to 0.15 microgram/ml) without interfering with the active antibody response to second and third doses of HbOC at 4 and 6 months of age.

Bacterial Capsules↗