Low prevalence of Epstein-Barr virus in incident gastric adenocarcinomas from the United Kingdom.

Epstein-Barr virus has been associated with a proportion of typical gastric adenocarcinomas. Here we report that the prevalence of Epstein-Barr virus in gastric adenocarcinomas from the United Kingdom is one of the lowest in the World. Gastric adenocarcinoma is another tumour whose association with Epstein-Barr virus varies with the population studied.

Antibody responses of cattle immunized with the Tf190 adhesin of Tritrichomonas foetus.

The antibody response patterns of cattle after subcutaneous and intranasal immunizations with adhesin Tf190 of Tritrichomonas foetus were investigated. Reactions of antibody from cattle parenterally immunized with Tf190 revealed antigen specificity and Tf190 sensitization in the majority of the animals, as determined by Western blotting. The results also demonstrated strong preimmune immunoglobulin G2 (IgG2) binding to T. foetus antigens not seen in IgG1 profiles. Subcutaneous injections of Tf190 resulted in significant (P < 0.05) increases in serum IgG1 and IgG2 titers over time, as determined by parasite specific enzyme-linked immunosorbent assay. Immune sera also significantly inhibited parasite adhesion to mammalian cell lines compared to the level of inhibition obtained with preimmune sera (P < 0.05). Intranasal immunization with Tf190 failed to produce measurable parasite-specific antibody in serum; however, this immunization route did result in significant (P < 0.05) increases in parasite-specific IgA titers in cervical mucus secretions from immunized animals that were more resistant to intravaginal challenge with T. foetus than controls. These results suggest that systemic immunization with Tf190 results in serum antibody production and antiparasitic adhesin antibodies. Additionally, the results of challenge experiments with intranasally immunized animals suggests that Tf190 primes protective immune responses that lead to lower rates of infection among these animals.

Antigen-specific T-cell responses in cattle immunized with antigens of Tritrichomonas foetus.

The cellular immune responses of cattle immunized with antigens of Tritrichomonas foetus were investigated. Subcutaneous injections of antigen preparations primed bovine peripheral blood mononuclear cells (PBMC) by 30 days of immunization as demonstrated by antigen-specific proliferation and by cytokine production upon antigen challenge of PBMC. Antigen-specific T-cells derived from PBMC responded by production of interferon (IFN)-gamma message detected by reverse transcriptase polymerase chain reaction, secreted IFN-gamma detected by enzyme-linked immunosorbent assay, and intracellular IFN-gamma detected by flow cytometry. Phenotypic analysis of PBMC responding in vitro to parasite antigen demonstrated a shift from a mixed CD4+, CD8+, gammadelta+, to predominantly CD4+, CD8-, gammadelta- phenotype in the Tf190-primed PBMC. In conclusion, systemic immunization of cattle with parasite antigen results in priming of bovine T-cells that are antigen specific and can produce an anamnestic IFN-gamma response to subsequent stimulation with antigens of T. foetus.

Evaluation of ewe and lamb immune response when ewes were supplemented with vitamin E.

Fifty-two Targhee twin-bearing ewes were used in a factorial arrangement of treatments to investigate the role of supplemental vitamin E (vit E); 0 (NE) vs 400 IU of vit E x ewe x (-1)d(-1) (E) and parainfluenza type 3 (PI3) vaccination; none (NP) vs PI3 vaccination (P) in immune function. Parainfluenza type 3 vaccination was used to evoke an immune response. Ewes receiving PI3 were vaccinated at 49 and 21 d before the expected lambing date. Ewes receiving vit E were orally dosed daily, 32 to 0 d before lambing. Blood was collected from ewes at the time of the initial PI3 vaccination and 4 h postpartum. Blood was collected from lambs (n = 104) at 3 d postpartum. Ewe and lamb sera were analyzed for anti-PI3 antibody titers, immunoglobulin G (IgG) titers, and vit E concentrations. Colostrum was collected 4 h postpartum and analyzed for IgG. The model for ewe and lamb analysis included the main effects of vit E and PI3, sex (lambs model only), and their interactions. No interactions were detected (P > 0.20) for any ewe or lamb variables. Serum anti-PI3 titers were greater (P < 0.01) in P ewes and their lambs than NP ewes and their lambs. Serum vit E concentrations were greater (P < 0.01) in E ewes and their lambs than NE ewes and their lambs. Colostral IgG titers and serum anti-PI3 titers did not differ (P > 0.20) between E and NE ewes. Serum IgG titers in E ewes and their lambs did not differ (P > 0.15) from IgG titers in NE ewes and their lambs. Lamb anti-PI3 titers did not differ (P = 0.76) between lambs reared by E and NE ewes. These results indicate that, although supplemental vit E to the ewe increased lamb serum vit E concentration, it had no effect on measures used in this study to assess humoral immunity in the ewe or passive immunity to the lamb.

Renal SNA as the primary mediator of slow oscillations in blood pressure during hemorrhage.

Blood pressure contains a distinct low-frequency oscillation often termed the Mayer wave. This oscillation is caused by the action of the sympathetic nervous system on the vasculature and results from time delays in the baroreflex feedback loop for the control of sympathetic nerve activity (SNA) in response to changes in blood pressure. In this study, we used bilateral renal denervation to test the hypothesis that it is SNA to the kidney that contributes a large portion of the vascular resistance associated with changes in the strength of the slow oscillation in blood pressure. In conscious rabbits, SNA and blood pressure were measured during hemorrhage (blood withdrawal at 1.35 ml. min(-1). kg(-1) for 20 min). Spectral analysis identified a strong increase in power at 0.3 Hz in SNA and blood pressure in the initial compensatory phase of hemorrhage before blood pressure started to fall. However, in a separate group of renal denervated rabbits, although the power of the 0.3-Hz oscillation under control conditions in blood pressure was similar, it was not altered during hemorrhage. Wavelet analysis revealed the development of low-frequency oscillations at 0.1 Hz in both intact and denervated animals. In conclusion, we propose that changes in the strength of the oscillation at 0.3 Hz in arterial pressure during hemorrhage are primarily mediated by sympathetic activity directed to the kidney.

Demonstration of Tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls.

Portions of penis and prepuce were collected from 24 bulls with current or recent Tritrichomonas foetus infection. Epididymides were collected from seven of the bulls, and seminal vesicles and prostate were collected from four. Following immunohistochemical staining with two monoclonal antibodies (34.7C4.4 and TF1.15) prepared against T. foetus surface antigens, trichomonads were identified in sections from 15 of the bulls. Organisms were most often located in penile crypts in the midshaft and caudal regions and less often in preputial crypts. Trichomonads were not observed in sections from other genitalia or in subepithelial tissue. T. foetus antigen, however, was present in the cytoplasm of some epithelial cells and the cytoplasm of some mononuclear cells in subepithelial lymphoid aggregates and follicles. Preputial smegma was collected from 16 T. foetus-infected bulls and from 16 control bulls with negative T. foetus cultures. Preputial antibody levels to TF1.17, a surface antigen of T. foetus, were determined by an enzyme-linked immunosorbent assay. Preputial secretions from infected bulls contained specific antibody of each isotype and subisotype tested. IgG1 responses were the greatest, IgM and IgA responses were approximately equal, and IgG2 responses were low. Each isotype and subisotype response in infected bulls was significantly greater than that in the controls. These results confirm previous speculation concerning anatomical sites of infection and suggest that parasite antigen can be taken up and processed locally, resulting in deposition of specific IgG1, IgG2, IgA, and IgM antibodies in the preputial cavity.

Low-frequency renal sympathetic nerve activity, arterial BP, stationary "1/f noise," and the baroreflex.

The object of this study is to quantify the very low frequency (i.e., <0.1 Hz) interactions between renal sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Six rats were instrumented for chronic recordings of SNA and ABP. Data were collected 24 h after surgery at 10 kHz for 2-5 h and subsequently compressed to a 1-kHz signal. The power spectra and ordinary coherence were calculated from data epochs up to 1 h in length. The very low frequency spectra for both variables were fitted to a constant times f (-beta). The peak magnitude squared of the coherence near 0.4 Hz was 0.82 +/- 0.08, but the apparent linear coherence fell off quickly at lower frequencies so that it was close to zero for frequencies <0.1 Hz. Moreover, at these low frequencies beta, as computed by a coarse grain spectral analysis, was significantly (P < 0.01) different for SNA (0.66 +/- 0.12) and ABP (1.12 +/- 0.14). Assuming that SNA and ABP are stationary time series, the results of our classical spectral analysis would indicate that SNA and ABP are not linearly correlated at frequencies with a period more than approximately 10 s. Accordingly, we tested for stationarity by computing the spectral coherence and found that SNA and ABP are not stationary "1/f noise" within the frequency range from 0.02 to 2.0 Hz. Rather the SNA exerts control over the cardiovascular system through intermittent bursts of activity. Such intermittent behavior can be modeled by nonlinear dynamics.

Purification and expression of the Tf190 adhesin in Tritrichomonas foetus.

Bovine trichomoniasis is a sexually transmitted disease caused by Tritrichomonas foetus and characterized by early embryo loss. The mechanism of this loss is not known, although the parasite is known to cause inflammation and to have the ability to kill host cells by a contact-dependent cytotoxic mechanism. Antibody specific for a 190,000-Da surface complex (Tf190) was previously shown to inhibit this adhesion. In this study we used immunoaffinity chromatography to purify Tf190 from T. foetus in order to analyze its composition and examine its expression. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Tf190 followed by silver staining revealed three components of Tf190. Western blotting and antibody-binding experiments showed that the 140- and 60-kDa bands were immunogenic. By using a battery of monoclonal antibodies (MAbs) periodate-sensitive epitopes were identified on Tf190, suggesting that these epitopes contained carbohydrate structures. Analyses of affinity-purified Tf190 by high-performance liquid chromatography and gas-liquid chromatography demonstrated the presence of the monosaccharides and lipids known to be prominent constituents of the lipophosphoglycan (LPG) of T. foetus. Flow cytometry experiments on several isolates of T. foetus with Tf190-specific antibodies revealed that Tf190 was present on subpopulations of all isolates but that not all epitopes were present on every isolate. This pattern of reactivities on the different parasite isolates was confirmed by Western blots of whole-parasite extracts probed with MAbs and antiserum. These results suggest that although variation in the expression of epitopes of Tf190 occurs in different strains of T. foetus, the Tf190 adhesion complex is widespread in different populations of the parasite. The data further suggest that immunogenic structures, important in the adhesion of T. foetus to mammalian cells, are located in the LPG-like component of Tf190.

Multifiber renal SNA recordings predict mean arterial blood pressure in unanesthetized rat.

The goal of this analysis was to quantify the relationship between renal sympathetic nerve activity (SNA) and mean arterial blood pressure (MAP). We previously recorded renal SNA and MAP in conscious rats during a stressful behavioral stimulus and during a nonstressful stimulus. We then formulated a set of two linear, first-order differential equations that uses our SNA recordings after a time delay (the input) to predict fluctuations in MAP (the output). Our model has four parameters: 1) the cardiovascular time constant T that characterizes the frequency response function between the effector elements controlled by the sympathetic nerves and the cardiovascular system (1-5 s); 2) the effector time constant Te determined by the coupling between the sympathetic nervous system and the effectors (0.0-0.6 s); 3) the efferent time delay tau e between a change in SNA and a change in MAP (0.4-0.6 s); and 4) a proportionality constant C between fluctuations in SNA and fluctuations in MAP (0.3-3.4 mmHg/nV). The parameters of the model were determined that minimize the residual error between the simulated time series and the actual data time series for a stressful stimulus. Then we tested the ability of the transfer function to predict the MAP response to a nonstressful stimulus. In five of seven rats tested, the model's predictions were good, with mean cross-correlation coefficients for the predicted trials between 0.62 and 0.83. We show that multifiber renal SNA recordings can reliably predict changes in MAP in the unanesthetized rat. Thus the overall sympathetic drive to the cardiovascular system is indexed by renal SNA, although the vasomotor effectors driven by renal SNA control only approximately 20% of the blood cow.

First-order differential-delay equation for the baroreflex predicts the 0.4-Hz blood pressure rhythm in rats.

We have described a 0.4-Hz rhythm in renal sympathetic nerve activity (SNA) that is tightly coupled to 0.4-Hz oscillations in blood pressure in the unanesthetized rat. In previous work, the relationship between SNA and fluctuations in mean arterial blood pressure (MAP) was described by a set of two first-order differential equations. We have now modified our earlier model to test the feasibility that the 0.4-Hz rhythm can be explained by the baroreflex without requiring a neural oscillator. In this baroreflex model, a linear feedback term replaces the sympathetic drive to the cardiovascular system. The time delay in the feedback loop is set equal to the time delay on the efferent side, approximately 0.5 s (as determined in the initial model), plus a time delay of 0.2 s on the afferent side for a total time delay of approximately 0.7 s. A stability analysis of this new model yields feedback resonant frequencies close to 0.4 Hz. Because of the time delay in the feedback loop, the proportional gain may not exceed a value on the order of 10 to maintain stability. The addition of a derivative feedback term increases the system's stability for a positive range of derivative gains. We conclude that the known physiological time delay for the sympathetic portion of the baroreflex can account for the observed 0.4-Hz rhythm in rat MAP and that the sensitivity of the baroreceptors to the rate of change in blood pressure, as well as average blood pressure, would enhance the natural stability of the baroreflex.

Immunohistochemical detection of Tritrichomonas foetus in formalin-fixed, paraffin-embedded sections of bovine placenta and fetal lung.

An immunohistochemical technique using a monoclonal antibody was evaluated as a diagnostic tool to specifically label Tritrichomonas foetus in formalin-fixed, paraffin-embedded sections of placenta and fetal lung from bovine abortions. Trichomonads were demonstrated in tissues from each of 12 abortions due to T. foetus and none of 15 abortions due to other or unidentified causes. Moderate to marked background staining occurred only in severely autolyzed tissues from T. foetus-infected fetuses. The antibody faintly labeled 1 of 3 other species of trichomonads (Trichomonas gallinae) but did not label other protozoa, bacteria, or fungi tested.

Genograms: a psychosocial assessment tool for hospice.

Genograms are a valuable and non-threatening evaluation tool for hospice patients and families. The genogram provides basic information about the family including the role of each member and the family dynamics. As the diagram is drawn, family life cycle issues and relationships between family members become evident. The genogram may go beyond the household to include supportive neighbors, friends, and community resources. Religious and spiritual support is also noted. The information is used to assess family needs and to provide interventions both before the death and during bereavement.

Analysis of adhesion and cytotoxicity of Tritrichomonas foetus to mammalian cells by use of monoclonal antibodies.

The relationship of Tritrichomonas foetus adhesion to mammalian cells and cytotoxicity to these targets was investigated. High-adherence and low-adherence lines of T. foetus, derived by repeated adhesion to HeLa cells, showed high and low cytotoxicity, respectively, to HeLa cells. When parasites were separated from targets by membranes (0.4-microns pore size), no cytotoxicity was detectable. Monoclonal antibodies elicited against T. foetus that lowered adhesion also lowered parasite-mediated cytotoxicity. Flow cytometry experiments revealed that the levels of an adhesion- and cytotoxicity-blocking antibody bound to the surface of high-adherence clones of T. foetus were higher than those in low-adherence clones. Western blots of parasite extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were probed with an anti-T. foetus antibody. A molecule with a molecular weight of approximately 190,000 composed of subunits with molecular weights of approximately 140,000 to 150,000 and approximately 65,000 was identified. Immunoprecipitation experiments with metabolically labeled T. foetus and the same antibody confirmed that similar subunits were synthesized by the parasite. These results indicate that adhesion of T. foetus to mammalian cells is an important step in cytotoxic damage of these targets and that a surface adhesin on the parasite is involved in the adhesion mechanism.

Cytotoxic and hemolytic effects of Tritrichomonas foetus on mammalian cells.

Geographically distinct lines of Tritrichomonas foetus were assayed for their ability to cause cytotoxicity in nucleated mammalian cells and lysis of bovine erythrocytes. T. foetus was highly cytotoxic toward a human cervical cell line (HeLa) and early bovine lymphosarcoma (BL-3) but displayed low levels of cytotoxicity against African green monkey kidney (Vero) cells. In addition to variation in the extent of cytotoxicity toward different targets, differences in the levels of cytotoxicity in the same nucleated target occurred with different parasite lines. Whole T. foetus, unfractionated whole-cell extracts, and parasite-conditioned medium (RPMI 1640 without serum) all caused lysis of bovine erythrocytes. Lytic activity in the conditioned medium was substantially reduced by repeated freezing and thawing or heating to 90 degrees C for 30 min. Damage of mammalian target cells by live T. foetus could be reduced by the presence of protease inhibitors; however, such inhibitors did not diminish the lytic effects of conditioned medium. These results suggested that proteolytic enzymes were necessary for the lytic mechanism of the live parasites but were not required once lytic factors were released into the parasite-conditioned medium. They further suggested that the lytic molecules were either proteins or had proteinaceous components.

Identification of Tritrichomonas foetus in sections of bovine placental tissue with monoclonal antibodies.

Sections of bovine placenta from cases of bovine trichomoniasis were examined for the presence of Tritrichomonas foetus by standard histological methods using phase-contrast microscopy and by indirect immunofluorescence assay (IFA) employing monoclonal antibodies (mAbs) specific for T. foetus. Parasites were identified readily in deparaffinized tissue up to 4 yr old by IFA with 2 mAbs previously shown to bind to the surface of living T. foetus. These results indicated that the IFA provided a rapid and specific method of identifying T. foetus in tissue sections as compared to standard histological methods.

Identification of antigens of Sarcocystis cruzi sporozoites, merozoites, and bradyzoites with monoclonal antibodies.

Sporozoites and culture-derived merozoites of Sarcocystis cruzi were used to elicit monoclonal antibodies (MAb's) in mice. Some of these antibodies reacted with the surface of live sporozoites and merozoites as determined by immunofluorescence. An array of similar antigens was identified in Western blots of sporozoites by both anti-merozoite MAb's and an anti-sporozoite MAb. At least 1 antigen in blots of bradyzoites was identified by anti-merozoite MAb's and a cluster of antigens was identified by an anti-sporozoite antibody. These results indicate that several surface epitopes of sporozoites and merozoites are shared with molecules of bradyzoites and that antigen patterns of molecules bearing these epitopes in 3 stages of Sarcocystis may be either distinct or similar.

Inhibition of penetration of cultured cells by Eimeria bovis sporozoites by monoclonal immunoglobulin G antibodies against the parasite surface protein P20.

Five monoclonal antibodies (MAbs) were partially characterized and tested for their ability to inhibit penetration of Madin-Darby bovine kidney (MDBK) cells by sporozoites of Eimeria bovis. By indirect fluorescent-antibody assays, all MAbs reacted with acetone-fixed sporozoites, but only two MAbs, EbS9 (immunoglobulin G1) and EbS11 (immunoglobulin G2a), localized specifically on the plasmalemma of live sporozoites. Two of the five MAbs also reacted with acetone-fixed first-generation merozoites of E. bovis; however, none of the MAbs reacted with live merozoites. Treatment of live sporozoites with EbS9 or EbS11 resulted in 79 and 73% decreases, respectively, in sporozoite penetration of MDBK cells. No significant differences in cell penetration occurred in MDBK cells inoculated with sporozoites that had been treated with the other three MAbs. Both EbS9 and EbS11 reacted in Western blots (immunoblots) of sporozoites with the same 20,000-relative-molecular-weight protein. The antigens against which these neutralizing MAbs react might be useful in immunizing against bovine coccidiosis.

In vitro development and antigen analysis of Sarcocystis.

During the past year considerable progress has been made in developing an in vitro system for growing large numbers of merozoites (the disease-causing stages) of Sarcocystis species that infect livestock. This system provides researchers with a means to study mechanisms of immunity and pathogenesis; and may eventually be used to develop effective diagnostic tests and vaccines against these economically important parasites. In this article, Carl Speer and Don Burgess discuss their in vitro system and show how preliminary antigen analysis has helped to deduce a possible mechanism for the vascular damage commonly observed in Sarcocystis infections.