Stable-isotope analysis of a deep-sea benthic-fish assemblage: evidence of an enriched benthic food web.

In this study, fishes and invertebrates collected from the continental slope (1000 m) of the eastern North Pacific Ocean were analysed using stable-isotope analysis (SIA). Resulting trophic positions (T(P) ) were compared to known diets and habitats from the literature. Dual isotope plots indicated that most species groups (invertebrates and fishes) sorted as expected along the carbon and nitrogen axes, with less intraspecific variability than interspecific variability. Results also indicated an isotopically distinct benthic and pelagic food web, as the benthic food web was more enriched in both nitrogen and carbon isotopes. Trophic positions from SIA supported this finding, resulting in the assignment of fishes to different trophic positions from those expected based on published dietary information. These differences can be explained largely by the habitat of the prey and the percentage of the diet that was scavenged. A mixing model estimated dietary contributions of prey similar to those of the known diet of Bathyraja trachura from stomach-content analysis (SCA). Linear regressions indicated that trophic positions calculated from SIA and SCA, when plotted against B. trachura total length for 32 individuals, exhibited similar variation and patterns. Only the T(P) from SCA yielded significant results (stomach content: P < 0·05, stable isotope: P > 0·05).

Results of the premature birth national need-gap study.

OBJECTIVE: Family-centered care is a standard of practice in neonatal intensive care units (NICUs). The purpose of the study was to assess successes and opportunities for improvement with parents' experiences and involvement in their premature infants' care in NICUs. STUDY DESIGN: Researchers' surveyed 502 parents whose children were currently < or =30 months old, had been born at a gestational age < or =36 weeks and had gone through or were currently in NICUs. RESULT: Most parents of premature infants were reasonably satisfied with the access, attention and information received from physicians and nurses in the NICU. However, approximately one-fourth were only moderately satisfied and nearly 10% were dissatisfied. CONCLUSION: While progress has been made in meeting the needs of parents in the NICU, more work needs to be carried out to improve family-centered care efforts. Specific attention should be given to providing more information and interaction opportunities for families, which may ultimately improve NICU outcomes.

Purging tumor cells from bone marrow by use of antibody and complement: a critical appraisal.

This review highlighted several problems associated with the use of antibody and complement in the elimination of tumor cells from bone marrow that was to be used for transplantation, and it discussed some of the difficulties encountered in developing this approach in model systems. These problems should be seriously considered by any clinician contemplating this method for bone marrow purging.

Complement-dependent cytotoxic antitumor antibody. II. Quantitative determination of cell-bound immunoglobulin M1.

A method for the quantitative determination of rabbit IgM bound to cell surfaces has been developed. This method was based on the ability of goat IgG specific for rabbit IgM heavy chains to bind 125I-labeled protein A when bound to the antigen. With the use of this technique the production of specific IgM antitumor antibodies in New Zealand White rabbits after immunization with guinea pig hepatoma cells line-1 and line-10 was followed. Differences in the production of IgM were observed between the different bleedings from rabbits immunized with line-1. No significant IgM antibody was produced following immunization of rabbits with line-10 tumor cells. This indirect method for determining IgM on the cell surfaces was objective, easy to perform, and detected complement-fixing and noncomplement-fixing antibodies. In addition, this technique could be applied to quantify other components on the cell surface for which a suitable specific antibody was available.

Detection of complement-dependent antibody to tumor cells in sera of strain-2 guinea pigs cured of their tumors by BCG Treatment.

Sera of strain-2 guinea pigs (cured of line-10 tumor by BCG therapy) were tested for complement-dependent, cytotoxic antibody. About 30% of the sera tested contained significant cytotoxic acitivity with the addition of human, but not syngeneic, complement. Using papain pretreated line-10 cells, we detected antibody in about 50% of the sera with syngeneic sera as the source of complement. Antibody to line-10 was also demonstrated in selected sera by indirect fluorescence and the C1 fixation and transfer test.

Evidence for an anticomplementary factor associated with human bone marrow cells.

Complement (C)-mediated lysis of antibody-sensitized sheep erythrocytes was inhibited by the addition of human bone marrow cells. The anticomplementary activity could be attributed to a soluble factor that was released from the bone marrow cells. This factor inhibited at an early stage in the C-cascade and showed the characteristics of a factor that accelerates decay of C2. The release of such a factor by bone marrow cells would present an obstacle to the use of antibody and C to purge tumor cells from bone marrow that is to be used for autologous transplantation.

Selective loss of expression of a tumor-associated antigen on a human leukemia cell line induced by treatment with monoclonal antibody and complement.

The sensitivity of a common acute lymphoblastic leukemia-associated antigen (cALLa)-positive, human leukemia pre-B-cell line to killing by antibody and complement was studied. A stable subpopulation was selected by its ability to survive four sequential treatments with excess monoclonal antibody (MoAb) directed against an Mr 24,000 glycoprotein associated with human leukemia cells and excess rabbit complement. Analysis of the antigen expression by individual cells within the parental and the selected cell populations was achieved by flow cytometry and demonstrated a marked decrease of the leukemia-associated antigen expression on individual cells within the selected subpopulation. These low-antigen-density cells were stable in subculture, and the immunoglobulin heavy-chain gene rearrangement of the parent population and the low-antigen-density subpopulation were identical, indicating that they were derived from a single cell source. The selection of this subpopulation was specific in that the expression of a second antigen recognized by a cALLa-specific MoAb was not affected. The presence of subpopulations of tumor cells with low levels of surface antigen expression that are resistant to killing upon addition of excess antibody and complement will prove to be an obstacle to the use of this approach to eliminate tumor cells from bone marrow that is to be used for autologous transplantation.

Relationship between the intracellular cyclic adenosine 3':5'-monophosphate level of tumor cells and their sensitivity to killing by antibody and complement.

Metabolic inhibitors which have been shown to increase the sensitivity of the guinea pig hepatoma, line 10, to antibody-guinea pig complement killing were tested for their effect on the intracellular cyclic adenosine 3':5'-monophosphate (cAMP) level of the line 10 cells. It was found that cells rendered sensitive to antibody-guinea pig complement killing following drug treatment showed a decrease in their intracellular cAMP levels. Increased susceptibility to lysis was always associated with decreased intracellular cAMP levels. Optimal doses of certain inhibitors effectively increased the sensitivity of the cells to complement-dependent lysis and decreased the intracellular cAMP levels. Other drugs which did not increase sensitivity of the cells to lysis also failed to decrease the intracellular cAMP levels. No experimental conditions were found in which intracellular cAMP levels were decreased without an increase in the susceptibility of the cells to antibody-guinea pig complement killing.

Effect of inhibiting DNA, RNA, and protein synthesis of tumor cells on their susceptibility to killing by antibody and complement.

A number of metabolic inhibitors and chemotherapeutic agents have been found to increase the sensitivity of a chemically induced guinea pig hepatoma (line 1) to killing by antibody and complement. We have investigated whether the mechanism whereby these drugs increase sensitivity to killing is attributable to their primary action of inhibiting DNA, RNA, or protein synthesis. Line 1 cells incubated for 1, 4, or 17 hr with actinomycin D (25 microng/ml), adriamycin (40 microng/ml), or puromycin (5 micron/ml) or with 5-fold lower concentrations of these drugs were maximally inhibited (greater than 90%) in their ability to synthesize DNA, RNA, and protein within 1 hr. However, only cells incubated for 17 hr with the high concentrations of drugs showed increased sensitivity to killing by antibody and complement. Line 1 cells incubated with high concentrations of these drugs of 17 hr, washed, and resuspended in drug-free medium recovered their resistance to killing by antibody and complement within 4 hr. These cells ever after culture for 24 hr in drug-free medium did not regain their ability to synthesize DNA, RNA, or protein. A similar lack of correlation between synthesis of these macromolecules and sensitivity to antibody-complement-mediated killing was observed after the cells were treated with physical agents that inhibit macromolecular synthesis. Both heat-treated and X-irradiated cells were inhibited in their ability to synthesize DNA, RNA, and protein immediately after treatment; however, only X-irradiated cells (6 and 16 hr postirradiation) were increased in their sensitivity to antibody-complement-mediated killing. Our data show that the ability of line 1 tumor cells to resist humoral immune attack does not depend solely on their ability to synthesize DNA, RNA, or protein.

Capturing host plasmin(ogen): a common mechanism for invasive pathogens?

Plasmin is a potent enzyme that can dissolve blood clots and degrade extracellular matrix proteins. A number of pathogenic bacteria produce plasminogen activators. Many of these organisms can also bind plasmin(ogen) to surface receptors and protect the active enzyme from physiological inhibition. Cell-surface localization of plasmin may be a common mechanism used by bacteria to facilitate movement through normal tissue barriers.

Characterization of a type II'o group A streptococcal immunoglobulin-binding protein.

The opacity factor positive M type 2 group A streptococcal isolate, A207, expresses a unique functional type II'o IgG-binding protein which reacts with all four human IgG subclasses and rabbit IgG. In order to determine the gene product or products responsible for this activity, three genes of the vir regulon from this isolate were cloned, expressed and analysed. The fcr A2 gene coded for a protein binding hyman IgG1, IgG2 and IgG4 but not IgG3. The enn2 gene coded for a protein reacting exclusively with human IgA, while the emmL2 gene product bound IgG1, IgG2, IgG3 and IgG4 as well as rabbit but not horse or pig IgG. The IgG3-binding activity of the EmmL2 protein was functionally indistinguishable from the Form 1 IgG3-binding activity present in heat extracts of group A isolate A207.

Regulation of the activation of C1 in serum.

C1 has been partially purified from serum and its ability to activate has been studied. The activation status of C1 was measured using a sensitive hemolytic assay which allowed the activation status of C1 in serum to be monitored. C1 did not activate in serum but would spontaneously activate when separated from certain other serum proteins, in particular, the C1-inhibitor protein. The activation of C1 was time and concn dependent, but addition of fully activated C1 did not affect either the rate or extent of activation. The activation of C1 could be inhibited reversibly by C1-inhibitor. This action of the C1-inhibitor was over and above its ability to regulate fully activated C1 by covalent bond formation with either the serine esterase of C1s or C1r. The results presented suggest that the C1-inhibitor plays a dual role in the regulation of C1 activation. The regulatory actions of C1-inhibitor would account for the presence of C1 in a zymogen form and for the episodic nature of complement consumption observed in individuals with genetic deficiencies of the C1-inhibitor protein.

Restriction in the lytic efficiency of complement of different erythrocyte targets: a re-examination of the activities of horse C8 and C9.

Differences in the lytic efficiency of different complement sources have frequently been observed. This effect has been shown to be related to both the species of the target erythrocyte and the species composition of terminal complement components within the 5b-9 membrane attack complex. The majority of studies have indicated that the source of C9 is critical in controlling the range of erythrocyte species that can be lysed efficiently. One exception to this general finding was the report by Lachmann et al., 1973 (Immunology 24, 135-145), using horse serum as a complement source. In that study, horse C8 rather than C9 was implicated as the critical component. In this study, we have re-examined this observation and find that the restricted hemolytic potential of horse complement correlates absolutely with the presence of horse C9. The reason for the differences between our findings and those of the earlier study are discussed.

Isolation and partial characterization of a type IV bacterial immunoglobulin binding protein.

A series of bovine G streptococcal isolates were screened for expression of type IV Fc binding proteins. An isolate expressing high levels of type IV binding proteins was selected and expanded by use of a colony selection technique. A variety of different extraction procedures were compared and the optimal solubilization procedure was found to be hot acid extraction of the bacteria. The extracted protein could be affinity purified on a column of immobilized rabbit IgG. The type IV Fc binding protein was found to be antigenically unrelated to the type I, II or III bacterial Fc binding proteins and displayed the lowest affinity and most limited range of species and subclass reactivity of any bacterial Fc binding protein thus far characterized.

Comparison of type III Fc receptors associated with group C and group G streptococci.

The type III Fc receptors present on or secreted by a series of group C and G streptococcal strains were studied. All strains capable of binding radiolabeled human IgG were shown to do so via an antigenically related Fc receptor. Treatment of any of the bacterial strains with papain or trypsin resulted in solubilization of Fc receptor activity. The pattern of Fc receptor activity recovered following enzyme treatment was not uniform. Differences were observed both between group C and G strains as well as within group C and G strains. Analysis of secreted Fc receptors indicated the presence of five molecular forms of Fc receptor. Each form was present at some level in the supernatant of every group C and G strain studied. The relative concn of each form of receptor secreted varied from strain to strain. The Fc receptor activity secreted by each strain demonstrated a similar affinity for the Fc region of human IgG and all were antigenically related. These results suggest that there is a family of closely related Fc receptors associated with group C and G streptococcal strains.

Characterization of a gene coding for a type IIo bacterial IgG-binding protein.

Two antigenic classes of non-immune IgG-binding proteins can be expressed by group A streptococci. One antigenic group of proteins is recognized by an antibody prepared against the product of a cloned fcrA gene (anti-FcRA). In this study, the immunogen used to prepare the antibody that defines the second antigenic class was shown to be the product of the emm-like (emmL) gene of M serotype 55 group A isolate, A928. The emmL55 gene expressed in E. coli produced an M(r) approximately 58,000 molecule which bound human IgG1, IgG2, IgG3 and IgG4, as well as horse, rabbit and pig IgG in a non-immune fashion. These properties are characteristic of the previously described type IIo IgG-binding protein isolated from this strain. In addition, the recombinant protein was reactive with human serum albumin and fibrinogen. The emmL 55 gene sequence was analysed and found to have the organization and sequence characteristics of a typical class I emm-like gene.

Interaction of bacterial Fc receptors with goat immunoglobulins.

The interaction of type I (staphylococcal protein A) and type III (streptococcal FcRc) bacterial Fc receptors with goat immunoglobulins has been studied. Staphylococcal protein A bound poorly to the majority of goat immunoglobulins at all pHs tested. There was some evidence that protein A bound IgG2 better than IgG1, particularly at pH 8 and above. One of 10 sera tested demonstrated a high level of reactivity with protein A and this was shown to correlate with the presence of a natural antibody to protein A. The streptococcal Fc receptors, FcRc, bound efficiently to all goat IgG and goat sera tested. Both goat IgG subclasses reacted efficiently with the FcRc between pH 6 and 8. Inhibition of binding of 125I-FcRc to immobilized goat IgG enabled levels of IgG in goat serum to be estimated. These results suggest the streptococcal FcRc will be of value as an immunochemical reagent in studies involving isolation and quantitation of goat immunoglobulins.

Identification of an amino acid signature sequence predictive of protein G-inhibitable IgG3-binding activity in group-A streptococcal IgG-binding proteins.

Sequence comparison of six known group-A streptococcal IgG-binding proteins, sharing the common property of protein G-inhibitable IgG3-binding-activity, identified a highly conserved 35-amino-acid (aa) sequence (74-100% similarity) within an EQ-rich central conserved core region of each protein. A search of aa sequence databases identified four additional proteins with > 50% similarity to this consensus sequence. All of these proteins demonstrated protein G-inhibitable IgG3-binding activity. Taken together, these results identify a signature sequence that predicts the presence of a protein G-inhibitable IgG3-binding domain(s) in group-A streptococcal IgG-binding proteins.

Detection of rheumatoid-like factors in serum of chickens immunized with bacterial immunoglobulin binding proteins.

A simple, rapid two-stage competitive binding radioimmunoassay is described for detecting rheumatoid factor-like antibodies in the serum of chickens immunized with bacterial immunoglobulin binding proteins. The assay could be adapted to determine the species specificity of any rheumatoid factor-like antibody by changing the species of immobilized IgG used. This assay has important practical implications for selecting suitable antibodies for detection of bacterial immunoglobulin binding proteins leaching from affinity columns in the presence of a large molar excess of IgG, and in determining the relationship between antibodies to bacterial IgG binding proteins and rheumatoid factor production.

A simple procedure to use whole serum as a source of either IgG- or IgM-specific antibody.

Absorption of rabbit antiserum to Forssman antigen with immobilized staphylococcal Protein A or concanavalin A selectively removed IgG or IgM antibodies, respectively. This absorption procedure was more rapid than ion exchange chromatography on DEAE cellulose or molecular sieving on Sephadex G-200 and gave a better yield of functionally purer antibody. This absorption method gave antiserum suitable for the preparation of either IgM or IgG sensitized sheep erythrocytes and should be of value for the large-scale preparation of indicator cells required for the study of complement action and for detection of specific receptors on cell surfaces.