Adenosine diphosphate platelet dysfunction on thromboelastogram is independently associated with increased morality in traumatic brain injury.

PURPOSE: The purpose of this study is to determine if adenosine diphosphate (ADP) platelet dysfunction on thromboelastogram (TEG) is associated with increased in-hospital mortality in patients with head trauma. The hypothesis is that ADP dysfunction is associated with increased mortality. METHODS: This retrospective review evaluated trauma patients admitted to a level 1 trauma center from February 2011 to October 2013 who received a TEG. Patients were included if the TEG was drawn within the first 24 h of admission and the head abbreviated injury score was greater than or equal to three. Patients were categorized as severe ADP dysfunction if the degree of ADP inhibition on TEG exceeded 60 %. RESULTS: A total of 90 patients were included (no ADP dysfunction n = 37; ADP dysfunction n = 53). Initial Glasgow Coma Scale [GCS (12 ± 4 vs. 11 ± 5; p = 0.26)] and use of pre-injury antiplatelet agents (30 vs. 28 %; p = 0.88) were similar. Patients with ADP dysfunction on TEG had a higher in-hospital mortality rate (8 vs. 32 %; p < 0.01). ADP dysfunction was independently associated with in-hospital mortality upon fixed logistic regression (OR 6.2; 95 % CI 1.2-33) while controlling for age, gender, hypotension, pre-injury antiplatelet agents, GCS and Injury Severity Score. CONCLUSION: ADP dysfunction on TEG is associated with increased mortality in patients with traumatic brain injury.

Explaining leptokurtic movement distributions: intrapopulation variation in boldness and exploration.

Leptokurtic distributions of movement distances observed in field-release studies, in which some individuals move long distances while most remain at or near their release point, are a common feature of mobile animals. However, because leptokurtosis is predicted to be transient in homogeneous populations, persistent leptokurtosis suggests a population heterogeneity. We found evidence for a heterogeneity that may generate persistent leptokurtosis. We tested individuals of the Trinidad killifish Rivulus hartii for boldness in a tank test and released them back into their native stream. Boldness in the tank test predicted distance moved in the field releases, even after effects of size and sex were removed. Further, data from a 19-mo mark-recapture study showed that individual growth correlated positively with movement in a predator-threatened river zone where the Rivulus population is spatially fragmented and dispersal is likely to be a hazardous activity. In contrast, no such correlation existed in a predator-absent zone where the population is unfragmented. These results show that a behavioral trait, not discernible from body size or sex, contributes to dispersal and that a component of fitness of surviving "dispersers" is elevated above that of "stayers," a fundamental assumption or prediction of many models of the evolution of dispersal through hazardous habitat.

Exercise, mobility and aging.

The elderly population is growing both in size and in proportion of the total population. The costs to the community of the elderly being in poor health are also growing proportionately. The beneficial effects of exercise on various physiological and psychological parameters in the elderly have been well established. The effects of exercise on the mobility and independence of the elderly are also of primary concern, their maintenance being an important exercise goal. Impaired balance and gait are the 2 most significant risk factors for limited mobility and falls in the elderly. It is important to understand the effects of aging and exercise on these risk factors.

Effect of recombinant bovine interleukin-1 beta in normal calves and in calves infected with bovine herpesvirus type 1.

Bovine herpesvirus-1 (BHV-1) is an important pathogen of respiratory infections in cattle. Its continuing importance lies in its ability to predispose infected hosts to bacterial infections. In this present study, we determined whether the immunoregulatory effects induced by interleukin-1 (IL-1) could stimulate appropriate host defense mechanisms to influence the course of BHV-1 infection in cattle. We first evaluated the effect of different doses (10-1000 ng/kg) of IL-1 in normal cattle. A single administration of IL-1 was able to induce a dose-dependent increase in polymorphonuclear (PMN) cells as well as monocytes in peripheral blood. The number of CD3+ lymphocytes and gamma/delta T cells in peripheral circulation decreased transiently in a dose-dependent manner. In the disease model, the effect of IL-1 administration (300 ng/kg) 24 h before, at the time of, and 24 h after the BHV-1 challenge was assessed. As a single therapeutic modality, IL-1 did not significantly reduce the establishment or progression of BHV-1-induced disease. Nevertheless, our results demonstrated that the significant modulation of diverse immune parameters did not exacerbate disease. Thus, the use of IL-1 as an adjunct therapy or as a vaccine adjuvant in cattle can be safely considered in situations where BHV-1 infection is likely to occur.

Regulation of bovine acute phase responses by recombinant interleukin-1 beta.

The acute phase response is a collection of physiologic changes initiated early in the inflammatory process. This response is comprised of both localized changes at the site of infection or injury and the initiation of systemic responses, such as the increase in production of acute phase proteins. Cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) play key roles in the regulation of acute phase response in the species studied to date. To better characterize the acute phase response of cattle, recombinant bovine (rBo). IL-1 beta was administered to cattle. A single administration of rBoIL-1 beta was able to induce a dose dependent increase in body temperature, circulating leukocytes, and serum haptoglobin and fibrinogen concentrations, as well as a decrease in plasma zinc concentration. Five daily administrations of rBoIL-1 beta resulted in heightened and prolonged elevations of haptoglobin and fibrinogen. In addition, alpha 1-acid glycoprotein levels were increased, a response not seen after a single administration of rBoIL-1 beta. These results indicate that IL-1 is an important regulator of the acute phase response in cattle.

BHV-1 glycoprotein 1 and recombinant interleukin 1 beta efficiently elicit mucosal IgA response.

The mucosal immune response to most soluble antigens administered directly to the mucosal system is low and requires a large amount of antigen and frequent vaccinations. In this study we tested whether immunizing cattle at a site which shares lymphatic drainage with the nasal mucosa could prime local mucosal immunity. We further tested whether recombinant bovine IL-1 beta (rBoIL-1 beta) could potentiate the induction of mucosal immunity. Animals were immunized subcutaneously at the base of the ear (s.e.) with recombinant bovine herpesvirus-1 (BHV-1) envelope glycoprotein I (gI) (35 micrograms animal-1) emulsified in incomplete Freund's adjuvant with or without rBoIL-1 beta (500 ng kg-1) followed by a second immunization 42 days later. Animals were challenged with virulent BHV-1 intranasally 42 days after the second immunization. Mucosal IgA from the nares was induced after only one immunization, and enhanced by boosting. rBoIL-1 beta treated animals had higher levels of BHV-1 specific nasal IgA (p < 0.01) and serum neutralizing antibody (p < 0.05). rBoIL-1 beta-treated animals also had increased numbers of surface IgA+ (p < 0.05) and IgG1+ (p < 0.001) B cells after in vitro antigen (gI) stimulation of peripheral blood lymphocytes suggesting that there was a greater expension of IgA+ and IgG1+ B cells in rBoIL-1 beta treated animals. When challenged with BHV-1, 3 of 4 animals in the gI+rBoIL-1 beta group were fully protected from viral replication in the nares, while only 1 of 4 animals receiving gI alone was protected.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal c-myc transformed macrophage cell lines. I. Heterogeneity in ability to process and present antigen.

Processing of proteins into immunogenic forms and their subsequent presentation to T cells are mediated by APC. Monocytes and macrophages have long been recognized as one of the APC types. However, little is known about whether functional heterogeneity in processing and presentation exist within the monocyte/macrophage population. Past difficulties in obtaining clonal representatives of these populations have limited investigations in this regard. The c-myc-containing retrovirus MRV, previously shown to immortalize murine macrophages, was used to generate a large panel of macrophage cell clones. Differences observed in cell surface antigen expression and morphology demonstrated phenotypic heterogeneity among these clones. Functional heterogeneity was also observed both before and after IFN-gamma and IL-4 stimulation. The clones differ in their capacity to present several nominal antigens to T cell hybridomas. When parallel variation in ability to present both a nominal antigen and a peptide representing the epitope for which a T cell hybridoma was specific was observed among the clones, this variation correlated with the levels of surface MHC class II antigen the clones expressed. In contrast, diversity in the ability to process and present certain nominal antigens among clones that all presented the corresponding antigenic peptide with similar efficiency did not appear to be due to differences in levels of surface MHC class II molecules. Our results suggest that the macrophage clones are heterogeneous in their ability to both process and present several antigens. The ability to obtain macrophage tissue culture cell lines displaying phenotypic and functional heterogeneity should allow insight into the impact of normal macrophage heterogeneity on the outcome of immune responses in vivo.

Immunopotentiation of bovine respiratory disease virus vaccines by interleukin-1 beta and interleukin-2.

Three experiments, using 85 crossbred beef calves, were conducted to evaluate the adjuvanticity of single, multiple, and combined doses of recombinant bovine IL-1 beta (rBoIL-1 beta) and recombinant bovine IL-2 (rBoIL-2), with a modified-live bovine herpesvirus-1/parainfluenza-3 (BHV-1/PI-3) virus vaccine and a killed bovine viral diarrhea (BVD) virus vaccine. Cytokines were administered intramuscularly at vaccination but at different injection sites. All cytokine treatments increased non-major histocompatibility complex (MHC)-restricted cytolytic capability of peripheral blood mononuclear cells (PBMC) against virus-infected target cells and serum neutralizing (SN) antibody titers to BHV-1 and BVD virus. Multiple, consecutive injections of rBoIL-2 generally showed the greatest adjuvant effect, and no additive effect was observed when rBoIL-1 beta and rBoIL-2 were administered together. In a challenge experiment, calves were vaccinated with a modified-live BHV-1/PI-3 vaccine and infected with BHV-1 on Day 21. Cytokine-treated calves had higher SN antibody titers to BHV-1 than did the control calves at the time of challenge. Calves that were administered rBoIL-2 on 5 consecutive days shed less BHV-1 and had the highest SN antibody titer to BHV-1 (Day 28). These data suggest that rBoIL-1 beta and rBoIL-2 may be useful immunoadjuvants for bovine respiratory disease virus vaccines.

Potentiation of antibiotic therapy for bovine mastitis by recombinant bovine interleukin-2.

Adjunct therapy with recombinant bovine interleukin-2 and antibiotics for Staphylococcus aureus IMI was investigated in an attempt to improve the therapy of antibiotics alone. Treatment of established S. aureus IMI with Na-cephapirin or Cefa-Lak produced average cures of 32.0 and 41.8%, respectively. When Na-cephapirin treatment was combined with recombinant bovine interleukin-2 at either 3.3 or 10 mg, the therapeutic efficacy was improved by an average of 20 to 30%. When Cefa-Lak treatment was combined with recombinant bovine interleukin-2 at 10 mg, the therapeutic efficacy was improved on average by 20%. Recombinant bovine interleukin-2, formulated in the excipient of the commercial Cefa-Lak, also improved the therapeutic efficacy by 16% compared with Cefa-Lak alone. Recombinant bovine interleukin-2, formulated in Cefa-Lak, maintained biological activity at room temperature for at least 21 d. After intramammary infusion of recombinant bovine interleukin-2, no biologically active interleukin-2 was detected in milk 48 h (four milkings) after administration. These data suggest that cytokines may be used as adjunct therapy with existing mastitis antibiotics or formulations of existing commercial products to improve the therapeutic efficacy.

Interleukin 2 treatment of Staphylococcus aureus mastitis.

A study was conducted in dairy cows to evaluate the efficacy of recombinant bovine interleukin 2 (rBoIL-2) as an adjunct to antibiotic therapy in Staphylococcus aureus mastitis. In normal, non-mastitic cows, intramammary infusion of rBoIL-2 caused a tenfold increase in somatic cell counts (SCC) in milk. Co-administration of 2 mg of rBoIL-2 and sodium cephapirin in cows with established S. aureus mastitis decreased SCC and shedding of S. aureus compared with values from cows that were given only sodium cephapirin or 10 mg rBoIL-2 with sodium cephapirin. Cows in the 2 mg rBoIL-2 group cleared the infection earlier and at 2 weeks after treatment had not relapsed with staphylococcal mastitis. These data suggest that rBoIL-2 may be useful as an immunotherapeutic agent in controlling mastitis.

Lysostaphin: immunogenicity of locally administered recombinant protein used in mastitis therapy.

A recombinant bactericidal protein, recombinant lysostaphin (r-lysostaphin), that may be useful as an intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle, was evaluated for immunogenicity to various hosts. Although immunogenicity could be demonstrated in a variety of other species when administered parenterally, oral administration failed to elicit a significant immunological response. Similarly, intramammary infusion of r-lysostaphin failed to elicit significant serum titers in the bovine until 18-21 infusions were administered (total administered dose of 2-3 g of protein). Antibody titers from dairy cattle which did develop an immune response were predominantly of the IgG1 subclass. Dairy cattle with significant anti-lysostaphin titers showed no deleterious symptoms (anaphylaxis, etc.) upon subsequent infusion, and these titers did not effect the in vitro bacteriostatic activity of r-lysostaphin. Intramammary infusion of r-lysostaphin does not elicit any observable effects on the host animal or on the potential efficacy of the recombinant molecule. Intramammary recombinant proteins may be suitable effective and safe infusion products that provide an alternative to classical antibiotic therapy.

Lysostaphin: use of a recombinant bactericidal enzyme as a mastitis therapeutic.

A recombinant mucolytic protein, lysostaphin, was evaluated as a potential intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle. Lysostaphin, a product of Staphylococcus simulans, enzymatically degrades the cell wall of Staphylococcus aureus and is bactericidal. Thirty Holstein-Freisian dairy cattle in their first lactation were infected with Staphylococcus aureus (Newbould 305, ATCC 29740) in all quarters. Infections were established and monitored for somatic cell counts and Staphylococcus aureus colony-forming units 3 wk prior to subsequent treatment. Infected animals were injected through the teat canal with a single dose of recombinant lysostaphin (dose response 1 to 500 mg) or after three successive p.m. milkings with 100 mg of recombinant lysostaphin in 60 ml of sterile phosphate-buffered saline. Animals were considered cured if the milk remained free of Staphylococcus aureus for a total of 28 milkings after last treatment. Kinetic analysis of immunologically active recombinant lysostaphin demonstrated that a minimum bactericidal concentration was maintained in the milk for up to 36 to 48 h after a single infusion of 100 mg of recombinant lysostaphin. The cure rate of quarters receiving recombinant lysostaphin (100 mg in sterile phosphate-buffered saline, administered over three consecutive p.m. milkings) was 20% compared with 29% for sodium cephapirin in saline and 57% for a commercial antibiotic formulation, respectively. An improved formulation of recombinant lysostaphin may prove to be an effective alternative to antibiotic therapy for bovine mastitis.

Staphylococcus aureus mastitis: pathogenesis and treatment with bovine interleukin-1 beta and interleukin-2.

Polymorphonuclear leukocytes play a central role in the pathogenesis of bovine mastitis. Intramammary challenge with Staphylococcus aureus was shown to induce both quantitative and qualitative changes in mammary gland polymorphonuclear leukocytes. Intramammary infusion of recombinant bovine interleukin-1 beta and interleukin-2 elicited a similar cellular response. Staphylococcus aureus, interleukin-1 beta, and interleukin-2 all increased the number of somatic cells after intramammary infusion and activated the inducible superoxide production in milk polymorphonuclear leukocytes. Interleukin-2 also activated phagocytosis of these cells, and their activation was maintained for 3 to 5 d after intramammary administration. Interleukin-1 beta and interleukin-2 were moderately effective in the therapy of experimental S. aureus mastitis. Approximately 54% of the glands treated with interleukin-1 beta responded to therapy by transiently clearing the milk of S. aureus, 30% of which relapsed, and a total of 38% of the treated glands remained cured. In contrast, 83% of glands treated with interleukin-1 beta responded to therapy, but 50% of these quarters relapsed. A total of 42% of the quarters treated with interleukin-1 beta remained cured. Homologous recombinant cytokines are effective immunomodulators that augment natural defensive mechanisms similar to the normal response to pathogens and may prove to be suitable alternatives to, or may be used in combination with, antibiotics as effective mastitis therapeutic agents.

Quantitative and qualitative properties of host polymorphonuclear cells during experimentally induced Staphylococcus aureus mastitis in cows.

Polymorphonuclear cells have a critical role in the pathogenesis of bovine mastitis. We have documented that experimentally induced Staphylococcus aureus mastitis is associated with cyclic increase and decrease in the quantity of viable bacteria shed in the milk. Concomitant with this cycling of bacteria is an inverse cycling of the hosts cells within the milk. Such somatic cells were determined to be greater than or equal to 95% polymorphonuclear cells. The quality of these cells was evaluated by measuring their relative efficiency of bacterial killing and phagocytosis at various times during an infection. Host polymorphonuclear cells had as much as 10,000-fold variation in the bactericidal failure rate for staphylococci during cell cycling. The most efficient bactericidal effect was observed at or near the peak of the somatic cell count (SCC). The ability of these cycling cells to ingest fluorescent beads was also quantitated by use of flow cytometry. The percentage of phagocytic polymorphonuclear cells that ingested fluorescent latex beads ranged from 15 to 80% of the total cell population during cell cycling, and tended to be optimal at or near peak SCC. In addition, the average number of beads ingested varied between 1 and 2 particles/polymorphonuclear cell, with as many as 17% of the phagocytic cells ingesting 4 or more beads at maximal efficiency. Polymorphonuclear cells from quarters infected with S aureus varied quantitatively (total SCC) and qualitatively (bactericidal activity and phagocytic ability) during the course of an infection. Not only is the quantity of host's phagocytic cells in the mammary gland central to the defense mechanism against infection, but the biological activation state appears to be equally important. The role of these cells in the pathogenesis of a cycling infection is presented in a model to explain the cyclic nature of mastitis.

Transplantation resistance to a murine plasmacytoma lacking MHC determinants.

A spontaneously arising murine plasmacytoma, HPC-202, derived from a BALB/c.H-2b congenic mouse that lacks any detectable H-2 determinants on its cell surface is described. However, the expression of H-2 determinants is inducible by interferon-gamma. The H-2 negative cell surface phenotype permits the HPC-202 tumor to escape H-2 allospecific cytotoxic cell lysis but not NK cell lysis, as well as to grow, to varying degrees, in some H-2 incompatible hosts. In those strains which exhibit a resistance to HPC-202 growth, resistance does not map to a single gene within the major histocompatibility complex of the mouse. Resistance is also radiosensitive and is therefore presumably due to a rapidly dividing cell population. The utility of this tumor as a model system to study both the non-H-2-restricted natural resistance to tumor growth, and the mechanism by which H-2 genes are regulated by cells is discussed.

Functional and biochemical characterization of a secreted I-J(+) suppressor factor that binds to immunoglobulin.

A secreted product of a T cell leukemic cell line, LH-8, was examined for its biochemical and biological properties. The factor that we have termed Immunoglobulin-Binding T cell Suppressor Factor (IgB-TsF) was shown to be suppressive for the in vitro and in vivo humoral response to a variety (but not all) antigens tested. The cell surface phenotype of the LH-8.1 subclone was M.Ig(-), Thy-1(+), L3T4(-), Lyt-2(+), FcR(-), MAC-1(-), and H-2b(+). In addition, both the cell surface and secreted factor, IgB-TsF, of LH-8.1 expressed determinants that were recognized by anti-I-Jb mAbs but not by an anti-I-Jd monoclonal. The same factor also retained an affinity for the Fc portion of approximately 30% of randomly selected, purified mAbs. This binding could be abolished if the Fab or F(ab')2 fragments of these mAb were used, but was found to be unrelated to isotype of the respective mAbs. Using subclones that expressed quantitative differences in their ability to exert suppression as sources of biosynthetically labeled IgB-TsF, we have shown the suppressor activity correlated with a single, 28 kD protein. Furthermore, comparisons of these same subclones that differ in their suppressor activity, do not show any direct correlation of this biological activity with the expression of the previously described T cell receptor genes. It also suggests that at least some suppressor cell subsets may use the same or related family of T cell receptor genes for their recognitive stage of activation as helper and cytotoxic T cell subsets, but not for their effector stage of immunologic suppression.

Expression of cell surface determinant(s) on murine myeloma stem cells and hematopoietic stem cells.

Murine B cell lymphomas and myelomas were examined for the expression of a determinant previously found exclusively on normal pluripotent stem cells colony-forming unit-spleen (CFU-s). This determinant(s), which is defined by a rabbit antimouse brain antiserum (R alpha MB), is present on the tumor stem cell population of some but not all B cell neoplasms examined. The determinant is not detected on tumor cells of the macrophage or T cell lineage. Absorption of the activity in R alpha MB with myeloma cells, concomitantly removed reactivity with the normal stem cell, CFU-s, and the myeloma stem cell, plasmacytoma CFU-s. Sorting analysis further showed that the antigen was diminished within a positive tumor population as cells acquired the capacity to secrete immunoglobulin. These studies suggest that this normal stem cell-associated antigen may also be an early differentiation antigen for the B cell lineage, and is expressed on some stem cells of B cell tumors.

Effect of IFN-gamma on the immune response in vivo and on gene expression in vitro.

T lymphocytes produce a variety of immunoregulatory molecules including gamma interferon (IFN-gamma) and antigen-specific suppressor and enhancer factors. During our studies of active substances obtained from cloned T-cell lines, we observed that certain fractions administered to mice resulted in enhancement of immune responses. Preliminary characterization of the substance suggested that it could be IFN-gamma and we therefore undertook a study of the action of IFN-gamma produced by recombinant DNA methodology on immune responses. We found that for several antigens, administration of IFN-gamma to mice leads to two- to five-fold enhancement of antibody formation provided that the IFN-gamma and antigen are administered together. The effect was dose dependent, giving a maximal response at 500-600 anti-viral units per mouse. Preliminary studies suggest that the macrophage may be the target of IFN-gamma action. Addition of IFN-gamma to cultures of a macrophage cell line leads to a greater than 10-fold increase in the level of RNA coding for I-region-encoded cell surface molecules.

Intratumor maturational heterogeneity within the murine myeloma MOPC-315.

The murine myeloma MOPC-315 secretes a paraprotein which binds dinitrophenylated compounds. Maturational subsets which mimic normal B-cell differentiation were shown to exist within this monoclonal neoplasm. The maturational subsets were defined by an in vivo stem cell activity (plasmacytoma colony-forming unit-spleen), DNA synthesis ([3H]thymidine incorporation), membrane-bound paraprotein, and cells secreting the MOPC-315 paraprotein. Velocity sedimentation at unit gravity separated cells enriched for secreting the MOPC0-315 protein (14.6 mm/hr) from the plasmacytoma colony-forming unit-spleen enriched population (9.3 mm/hr). An in vivo sequential analysis of the appearance of tumor (i.v. challenge) in the spleen of BALB/c hosts did not reveal any discordant appearance of these same maturational subsets with respect to time. The MOPC-315 myeloma contains maturational subsets that mimic normal B-cell differentiation and apparently differentiates during a very early stage of its evolution within the host.