A novel IL-18BP ELISA shows elevated serum IL-18BP in sepsis and extensive decrease of free IL-18.

IL-18 binding protein (IL-18BP) is a circulating antagonist of the proinflammatory Th1 cytokine IL-18. It effectively blocks IL-18 by forming a 1:1 high affinity (Kd=400 pM) complex, exhibiting a very low dissociation rate. We have developed a sandwich ELISA for IL-18BPa and determined its limit of detection (62 pg/ml). Interference by IL-18 and related cytokines, as well as cross reactivity with other IL-18BP isoforms (b, c, and d) were determined. Using this ELISA, we measured serum IL-18BPa in large cohorts of healthy individuals and in septic patients. Serum IL-18BPa in healthy individuals was 2.15+/-0.15 ng/ml (range 0.5-7 ng/ml). In sepsis, the level rose to 21.9+/-1.44 ng/ml (range 4-132 ng/ml). Total IL-18 was measured in the same sera by an electrochemiluminescence assay and free IL-18 was calculated based on the mass action law. Total IL-18 was low in healthy individuals (64+/-17 pg/ml) and most of it ( approximately 85%) was in its free form. Total IL-18 and IL-18BPa were both elevated in sepsis patients upon admission (1.5+/-0.4 ng/ml and 28.6+/-4.5 ng/ml, respectively). At these levels, most of the IL-18 is bound to IL-18BPa, however the remaining free IL-18 is still higher than in healthy individuals. We conclude that IL-18BPa considerably inhibits circulating IL-18 in sepsis. Yet, exogenous administration of IL-18BPa may further reduce circulating IL-18 activity.

Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads.

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.

Relationship between resistance to complement, virulence and outer membrane protein patterns in pathogenic Escherichia coli O2 isolates.

To establish a possible relationship between resistance to complement, virulence and outer membrane protein banding patterns, ten E. coli O2 strains isolated from chickens with colibacillosis were studied for: (1) resistance to the bactericidal effect of complement by a quantitative microtiter method, (2) virulence, as determined by chicken lethality test, and (3) outer membrane protein banding patterns yielded by SDS-polyacrylamide gel electrophoresis. The ten isolates were classified into three groups: (1) Group 1, consisting of four isolates showed: (a) high resistance to complement, (b) high virulence, and (c) different pattern between 35 and 40 kDa with a weak peptide band at 35 kDa. (2) Group 2, consisting of one isolate showed: (a) high resistance to complement, (b) low virulence, and (c) a weak peptide band at 35 kDa. (3) Group 3, consisting of five isolates showed: (a) low resistance to complement, (b) low virulence, and (c) identical OMP pattern between 35 and 40 kDa exhibiting a strong peptide band at 35 kDa. The results suggest that high resistance to complement may be necessary but no sufficient for virulence and that OMP banding patterns may be a marker for virulence.

Tumor-preventive effects of the soluble p53 antigen on chemically-induced skin cancer in mice.

BACKGROUND: The tumor-suppressive effects of the rat soluble p53 antigen on chemically induced skin cancer in mice and the role of the spleen in the immune response to a carcinogen and vaccination were studied. METHODS: Skin cancer was induced by 9,10-dimethyl-1,2-benzanthracene (DMBA). Vaccination was initiated by injection of liposomes with the soluble p53 antigen (10-12 micrograms/mouse) while boosters were with the p53 mixed with Freund's incomplete adjuvant (two injections). Four months later, the spleen and tumors were removed and examined morphometrically (determination of areas of different spleen's zones) and immunohistochemically (determination of number of B lymphocytes and macrophages, apoptotic index). The following groups of mice were studied: A) control non treated mice; Bl) tumor-free mice treated with a carcinogen; B2) tumor-bearing mice; Cl) tumor-free vaccinated mice exposed to a carcinogen; C2) tumor-bearing vaccinated mice. RESULTS: Mice exposed to a carcinogen, which were tumor-free, displayed high proliferative activity of the spleenic lymphoid constitutes such as B lymphocytes and macrophages. This was reflected in the remarkable transformation of B lymphocytes in lymphoblasts (blast transformation) and an increase in the area of germinal centers, compared to untreated controls. In tumor-bearing non vaccinated mice, significantly more spleenic apoptotic cells were found than in their tumor-free counterparts. Shrinkage of the mantle layer and a decrease in cellular density of follicles were seen in all carcinogen-treated mice, reflecting the reduced total production of lymphoid cells, and thus the insufficiency of the immune reaction of animals to a carcinogen. A sharp decrease in the apoptotic index in the spleen of tumor-free mice may reflect an inhibition of apoptotic activity of the spleen by a carcinogen. Vaccination with the soluble p53 protein decreased the incidence of tumors and their size, significantly increased the apoptotic index within tumors, and reversed the splenic parameters of immune insufficiency. CONCLUSIONS: The immune system is active during tumorigenesis. Vaccination with the soluble p53 antigen had positive tumor-suppressive effects. The findings may facilitate the development of vaccines for the prevention of recurrent cancers in humans.

Chicken-embryo fibroblasts produce two types of interferon upon stimulation with Newcastle disease virus.

Controversy has long surrounded the question of whether chickens, like mammals, can produce two types of interferon (IFN). Recently, type-I and type-II chicken IFNs have been cloned. Our study focuses on the further characterization of native fibroblast and spleen IFNs and shows that chicken embryo fibroblasts (CEFs) produce a mixture of type-I and type-II IFNs. IFN was purified by three different methods, controlled pore-glass chromatography, ion-exchange chromatography and preparative SDS-PAGE. Three protein bands showing IFN-like anti-viral activity, from CEFs which had been virus-stimulated for IFN production, were detected at 25, 27 and 29 kDa. Polyclonal antibodies produced against these bands showed partial cross-reaction with purified media from mitogen-stimulated spleen cells in ELISA, western blot analysis and anti-viral activity neutralization assay. Differences between purified conditioned media from CEF and spleen were found with respect to the stimulation of macrophages for nitric oxide production, pH stability and signal transduction pathways; only CEF IFN activated the IFN-stimulated gene factor-3 complex, whereas both CEF and spleen IFNs activated the IFN regulatory factor-1 gene. These findings concur with the differences that are known to exist between mammalian type-I and type-II IFNs. Attempts at sequencing the 25 and 27 kDa proteins by Edman degradation yielded evidence of N-terminal blockage.

Vaccination of turkey poults against pathogenic Escherichia coli.

A vaccine made of bacterial membranes which form vesicles has been developed in our laboratory and found to be effective in protecting chickens against challenge with pathogenic E. coli. In the present study we monitored maternal anti-5. coli antibody in turkey poults and found that although at hatch the antibody titre was relatively high, it decreased gradually to a low level by 14 days of age and remained low up to 31 days of age. Both intramuscular and subcutaneous vaccination with membrane vesicles (MV) resulted in increased antibody titres. Poults with high antibody titres could survive challenge with the pathogenic bacteria. Serum from vaccinated poults reduced E. coli bacterial growth rate when added to the growth medium at a high concentration, whereas serum from non-vaccinated poults did not. Lymphocytes from vaccinated poults responded with an increased rate of blastogenesis when MV was added to the cultures. Lymphocytes from non-vaccinated poults were not stimulated to blastogenesis. We concluded that MV can be used as a vaccine to render poults resistant to E. coli by evoking antibody production, stimulating the bacterial-lysis activity of complement, stimulating T cells to proliferate and stimulating cytotoxic T cells involved in disease resistance.

The serotyping of Azospirillum spp. by cell-gold immunoblotting.

Ten Azospirillum strains were serotyped using the method of cell-gold immunoblotting (dot-blot immune-overlay assay). Colloidal gold-protein A conjugate was used. Antibodies raised against the whole cells showed strain specificity and interacted mainly with carbohydrate antigens on the cell surface. Immunological identity for A. brasilense Sp 245 and Sp 107 strains was found. Cell-gold immunoblotting can be recommended for serotyping of a wide variety of bacterial strains.