Dynamics of bovine spleen cell populations during the acute response to Babesia bovis infection: an immunohistological study.

The spleen is a critical organ in defence against haemoparasitic diseases like babesiosis. Many in vitro and ex vivo studies have identified splenic cells working in concert to activate mechanisms required for successful resolution of infection. The techniques used in those studies, however, remove cells from the anatomical context in which cell interaction and trafficking take place. In this study, an immunohistological approach was used to monitor the splenic distribution of defined cells during the acute response of naïve calves to Babesia bovis infection. Splenomegaly was characterized by disproportionate hyperplasia of large versus small leucocytes and altered distribution of several cell types thought to be important in mounting an effective immune response. In particular, the results suggest that the initial crosstalk between NK cells and immature dendritic cells occurs within the marginal zone and that immature dendritic cells are first redirected to encounter pathogens as they enter the spleen and then mature as they process antigen and migrate to T-cell-rich areas. The results of this study are remarkably similar to those observed in a mouse model of malarial infection, suggesting these dynamic events may be central to the acute response of naïve animals to haemoparasitic infection.

The bovine spleen: interactions among splenic cell populations in the innate immunologic control of hemoparasitic infections.

Over the past several years, innate immunity has been recognized as having an important role as a front-line defense mechanism and as an integral part of the adaptive immune response. Innate immunity in cattle exposed to hemoparasites is spleen-dependent and age-related. In this review, we discuss general aspects of innate immunity and the cells involved in this aspect of the response to infection. We also provide examples of specific splenic regulatory and effector mechanisms involved in the response to Babesia bovis, an important tick-borne hemoparasitic disease of cattle. Evidence for the regulatory and effector role of bovine splenic monocytes and DC both in directing a type-1 response through interaction with splenic NK cells and γδT-cells will be presented.

Physical activity and physiological cardiac remodelling in a community setting: the Multi-Ethnic Study of Atherosclerosis (MESA).

OBJECTIVE: To evaluate the association of physical activity with left ventricular structure and function in the general population in a community setting. DESIGN: Cross-sectional study. SETTING: The Multi-Ethnic Study of Atherosclerosis (MESA), a population-based study of subclinical atherosclerosis. PARTICIPANTS: A multiethnic sample of 4992 participants (aged 45-84 years; 52% female) free of clinically apparent cardiovascular disease. INTERVENTIONS: Physical activity induces beneficial physiological cardiac remodelling in a cross-sectional study of non-athlete individuals. MAIN OUTCOME MEASURES: Left ventricular mass, volumes and function were assessed by cardiac magnetic resonance imaging. Physical activity, defined as intentional exercise and total moderate and vigorous physical activity, was assessed by a standard semiquantitative questionnaire. RESULTS: Left ventricular mass and end-diastolic volume were positively associated with physical activity (eg, 1.4 g/m(2) (women) and 3.1 g/m(2) (men) greater left ventricular mass in the highest category of intentional exercise compared with individuals reporting no intentional exercise; p = 0.05 and p<0.001, respectively). Relationships were non-linear, with stronger positive associations at lower levels of physical activity (test for non-linearity; p = 0.02 and p = 0.03, respectively). Cardiac output and ejection fraction were unchanged with increased physical activity levels. Resting heart rate was lower in women and men with higher physical activity levels (eg, -2.6 beats/minute lower resting heart rate in the highest category of intentional exercise compared with individuals reporting no intentional exercise; p<0.001). CONCLUSIONS: In a community-based population free of clinically apparent cardiovascular disease, higher physical activity levels were associated with proportionally greater left ventricular mass and end-diastolic volume and lower resting heart rate.

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells.

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.

Differential response of splenic monocytes and DC from cattle to microbial stimulation with Mycobacterium bovis BCG and Babesia bovis merozoites.

Both bovine peripheral blood monocyte-derived dendritic cells (DC) and myeloid DC from afferent lymph have been described, but resident DC from other bovine tissues have not been fully characterized. The spleen as a secondary lymphoid organ is central to the innate and acquired immune response to various diseases particularly hemoprotozoan infections like babesiosis. Therefore, we developed methods to demonstrate the presence of myeloid DC from the spleen of cattle and have partially characterized a DC population as well as another myeloid cell population with monocyte characteristics. The phenotypic profile of each population was CD13+CD172a+/-CD14-CD11a-CD11b+/-CD11c+ and CD172a+CD13+/-CD14+CD11a-CD11b+/-CD11c+, respectively. The CD13+ population was found exclusively in the spleen whereas the CD172a+ population was present at the same percentage in the spleen and peripheral blood. CD13+ cells developed a typical veiled appearance when in culture for 96 h. The two cell populations differed in their ability to produce nitric oxide and had a different pattern of cytokine mRNA when stimulated with Mycobacterium bovis BCG or Babesia bovis merozoites. The data demonstrate the presence of a myeloid splenic DC with attributes consistent with an immature status.

The timed walk test as a measure of severity and survival in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive disease with a median survival of approximately 3 yrs. Measurements of airflow and lung volumes at rest are generally used to monitor the clinical course in this disorder. This study was designed to determine if a modified version of the 6-min walk test, called the timed walk test, accurately characterises disease severity and survival in IPF. The study population consisted of 28 patients with well-characterised progressive IPF. The timed walk test and concurrent measures of disease severity were assessed at baseline. Participants were prospectively followed for > or =4 yrs to determine the relationship between parameters of the timed walk test and survival. There were strong correlations between the end-exercise saturation and walk-velocity parameters of the timed walk test and diffusing capacity, and arterial oxygen tension at rest. In univariate Cox proportional-hazards models, end-exercise saturation, change in saturation with exercise, walk distance and walk velocity were associated with survival. In unadjusted logistic regression models, odds of death at 2 yrs were associated with the same parameters. In conclusion, the timed walk test relates to disease severity and long-term outcome in progressive idiopathic pulmonary fibrosis.

The innate immune response in calves to Boophilus microplus tick transmitted Babesia bovis involves type-1 cytokine induction and NK-like cells in the spleen.

The innate immune response to Babesia bovis infection in cattle is age-related, spleen-dependent and, in stabilate inoculated calves, has type-1 characteristics, including the early induction of IL-12 and IFN-gamma. In this study with three calves, parameters of innate immunity were followed for 2 weeks after tick transmission of B. bovis. Each calf survived the acute disease episode without drug intervention, and responded with increased levels of plasma interferon-gamma and type-1 cytokine expression, monocyte/macrophage activation, and CD8+ cellular proliferation in the spleen. The proliferating CD8+ population consisted primarily of NK-like cells, and the expansion occurred in parallel with an increase in IL-15 mRNA expression in the spleen.

Influence of herbicide tolerant soybean production systems on insect pest populations and pest-induced crop damage.

Conventional soybean weed management and transgenic herbicide-tolerant management were examined to assess their effects on soybean insect pest populations in south Georgia in 1997 and 1998. Soybean variety had very little impact on the insect species observed, except that maturity group effects were observed for stink bug, primarily Nezara viridula (L.), population densities on some sampling dates. Stink bugs were more abundant on the early maturing varieties in mid-season. Velvetbean caterpillar, Anticarsia gemmatalis Hübner, potato leafhopper, Empoasca fabae (Harris), and grasshoppers Melanoplus spp. were more numerous on either conventional or herbicide-tolerant varieties on certain dates, although these differences were not consistent throughout the season. Soybean looper, Pseudoplusia includens (Walker), threecornered alfalfa hopper, Spissistilus festinus (Say), and whitefringed beetles, Graphognathus spp , demonstrated no varietal preference in this study. Few weed treatment differences were observed, but if present on certain sampling dates, then pest numbers were higher in plots where weeds were reduced (either postemergence herbicides or preplant herbicide plus postemergence herbicide). The exception to this weed treatment effect was grasshoppers, which were more numerous in weedy plots when differences were present. In post emergence herbicide plots, there were no differences in insect pest densities between the conventional herbicides (e.g., Classic, Select, Cobra, and Storm) compared with specific gene-inserted herbicide-tolerant materials (i.e., Roundup and Liberty). Defoliation, primarily by velvetbean caterpillar, was different between soybean varieties at some test sites but not different between herbicide treatments. We did not observe differences in seasonal abundance of arthropod pests between conventional and transgenic herbicide-tolerant soybean.

Resistance of Geneva and Other Apple Rootstocks to Erwinia amylovora.

When vigorously growing shoots of 49 different apple rootstocks grown in a greenhouse were inoculated with different strains of Erwinia amylovora, Budagovsky 9 (B.9), Ottawa 3, Malling 9, and Malling 26 were the most fire blight susceptible rootstocks and Geneva 11, Geneva 65, Geneva 16, Geneva 30, Pillnitzer Au51-11, Malling 7, and several breeding selections were the most resistant. Significant strain-rootstock interactions were observed in the amount of fire blight that resulted from inoculation. Field-grown fruiting 'Royal Gala' trees on Geneva 16 and Geneva 30 rootstocks were highly resistant to rootstock infection (no tree mortality) when trees sustained severe blossom infection with E. amylovora, compared with Malling 9 and Malling 26 rootstock clones, which were highly susceptible to infection (36 to 100% tree mortality). In contrast to potted own-rooted B.9 plants inoculated in a greenhouse, B.9 rootstocks of orchard trees appeared resistant to rootstock infection (0% tree mortality). Orchard trees on Geneva 11 were moderately resistant to rootstock infection (25% tree mortality). There was general agreement in the evaluation of resistance under orchard conditions when rootstock resistance was evaluated in relation to controlled blossom inoculation or to natural blossom infection.

Age-related innate immune response in calves to Babesia bovis involves IL-12 induction and IL-10 modulation.

There is a strong innate immunity in calves to infection with Babesia bovis. Interleukin (IL)-12 and IL-10 have been shown in vitro to be important immunoregulatory cytokines. Here we demonstrate in vivo that the protective innate response in young calves to infection with virulent B. bovis involves the early appearance of IL-12 and interferon-gamma (IFN-gamma) transcripts in the spleen. In contrast, IL-12 and IFN-gamma mRNA expression in the spleens of adult cattle that succumbed to the infection was delayed and depressed and occurred within the context of IL-10 expression. Also in contrast with calves, there was no detectable antibody response before death in adults. A vigorous CD8+ T-cell expansion occurred in the spleens of both calves and adults.

IL-4 and IL-10 inhibition of IFN-gamma- and TNF-alpha-dependent nitric oxide production from bovine mononuclear phagocytes exposed to Babesia bovis merozoites.

The requirement for IFN-gamma and/or TNF-alpha as co-stimulants with Babesia bovis merozoites for nitric oxide (NO) production was examined, as well as the regulatory role of IL-4 and IL-10. Purified B. bovis merozoites did not induce the production of NO in undifferentiated monocytes without addition of exogenous IFN-gamma and TNF-alpha unless the monocytes taken ex vivo were producing TNF-alpha endogenously. Under the latter condition, the NO production resulting from merozoite stimulation remained IFN-gamma-dependent. There was no evidence for endogenous synthesis of TNF-alpha in monocyte-derived macrophages (MDM), and merozoites alone were incapable of inducing TNF-alpha mRNA in MDM. However, while merozoites plus IFN-gamma induced TNF-alpha mRNA expression in MDM, NO was not produced. Both IL-4 and IL-10 inhibited expression of iNOS and production of NO in merozoite-stimulated monocytes.

The age-related immunity in cattle to Babesia bovis infection involves the rapid induction of interleukin-12, interferon-gamma and inducible nitric oxide synthase mRNA expression in the spleen.

Young calves possess a strong innate immunity against Babesia bovis infection that lasts for approximately 6 months after birth and is abrogated with the removal of the spleen. This immunity is characterized as cellular involving a soluble mediator. Nitric oxide has been implicated by virtue of its babesiacidal affects in vitro, but questioned to be as effective in vivo, due to its ability to downregulate type-1 immunity. Spleen cells were obtained from 4-month-old calves and adult steers and processed for monitoring cytokine and inducible nitric oxide synthase (iNOS) mRNA expression during the response to initial B. bovis infection. The data provided evidence of a transient role for nitric oxide in innate immunity, characterized by brief iNOS induction in the spleen of calves that was not detectable in the spleens of adults. The iNOS message followed the early induction of interleukin (IL)-12 and interferon (IFN)-gamma message in calves. The induction of IL-12 and IFN-gamma message in adults was delayed until IL-10 message was induced. Transformation growth factor-beta mRNA expression levels were greater in spleen cells from adults early in infection and then declined, whereas expression levels increased in spleen cells from calves later in the infection process. Together, the data support the concept of 'first come, first serve' cytokine influence over cellular activities, the importance of a type-1 response in the control of an initial infection and the need for tight regulation in order to prevent pathology associated with over production of nitric oxide and inflammatory cytokines.

Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi infections of Moroccan origin and its use in determining the seroprevalence of B. equi in Morocco.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay for detection of specific antibody to Babesia equi in serum from equids was validated for use in Morocco. The assay is based on the specific inhibition of binding of a monoclonal antibody to a conserved epitope within a recombinant parasite peptide by serum from infected animals. The assay was compared to an established indirect immunofluorescence assay, with a concordance of 91%. The assay was used to determine seroprevalence for B. equi infections in donkeys and horses throughout Morocco. A total of 578 sera (163 horses and 415 donkeys) from 6 locations representing different bioclimatic regions were assayed. An analysis of variance, indicated no significant effect of location; however, donkeys were significantly more likely than horses to be seropositive. Management conditions contribute to greater tick infestations and thus Babesia exposure in donkeys than in horses.

Effective and safe modification of multiple atherosclerotic risk factors in patients with peripheral arterial disease.

BACKGROUND: Patients with peripheral arterial disease (PAD) are at an increased risk of cardiovascular mortality and morbidity and thus are an excellent group in whom to evaluate the feasibility and the effect of an aggressive multifactorial intervention on atherosclerotic vascular disease risk factors. The Arterial Disease Multiple Intervention Trial (ADMIT) was designed to determine the efficacy, safety, and compliance of an multifactorial therapy on selected atherosclerotic disease risk factors in patients with PAD. METHODS: By a 2 x 2 x 2 factorial design, eligible participants (N = 468) were randomly assigned to low-dose warfarin, antioxidant vitamins, and niacin or its corresponding placebo, and followed up for 1 year. All participants were encouraged to use aspirin. Pravastatin was added to the drug regimen for those who needed to reduce LDL cholesterol to recommended levels. RESULTS: Niacin increased HDL cholesterol levels by 30%, with the majority of effect achieved at a dosage of 500 mg twice daily. Warfarin had an anticoagulant effect. The antioxidant vitamins resulted in a significant increase in vitamin E, C, and beta-carotene plasma levels. Overall, compliance was high and few adverse effects were reported. CONCLUSIONS: ADMIT demonstrates that it is both feasible and safe to modify multiple atherosclerotic disease risk factors effectively with intensive combination therapy in patients with PAD.

A comparative evaluation of polytetrafluoroethylene, umbilical vein, and saphenous vein bypass grafts for femoral-popliteal above-knee revascularization: a prospective randomized Department of Veterans Affairs cooperative study.

PURPOSE: Currently, the choice of a vascular prosthesis for a femoral-popliteal above-knee arterial bypass graft is left to the surgeon's preference, because the available information on comparative evaluations is inconclusive. The Department of Veterans Affairs (VA) Cooperative Study 141 was established to identify whether improved patency exists with different bypass graft materials for patients with femoral-popliteal above-knee bypass grafts. METHODS: Between June 1983 and June 1988, 752 patients at 20 VA medical centers were randomized to receive either an externally supported polytetrafluoroethylene (PTFE; N = 265), human umbilical vein (HUV; N = 261), or saphenous vein (SV; N = 226) for an above-knee femoral-popliteal bypass graft. The indication for the bypass grafting operation was limb salvage in 67.5% of the patients. Patients were observed every 3 months for the first year and every 6 months thereafter. All patients were instructed to take aspirin (650 mg) daily for the duration of the study.Doppler-derived ankle-brachial indices (ABIs) were determined preoperatively and serially postoperatively. A bypass graft was considered to be patent when the Doppler-derived postoperative ABI remained significantly improved (more than 0.15 units higher than their preoperative value) and additional objective information, such as angiograms or operations, did not contradict these observations. Patency failure also included bypass grafts that were removed because of an infection or aneurysmal degeneration. Patency rates were compared by using the Kaplan-Meier life table analysis. RESULTS: The cumulative assisted primary patency rates were statistically similar among the different conduit types at 2 years (SV, 81%; HUV, 70%; PTFE, 69%). After 5 years, above-knee SV bypass grafts had a significantly (P

The relative order of helical propensity of amino acids changes with solvent environment.

A model peptide of sequence Ac-Y-VAXAK-VAXAK-VAXAK-NH(2), where X is substituted with one of nineteen amino acids (P excluded), was synthesized and titrated with methanol to study helical propensity as a function of solvent environment. The CD spectra of these peptides are largely random coil in 2 mM sodium phosphate buffer (pH 5.5) and show a conformational change to alpha-helix with increasing methanol content. Singular value decomposition was used to correct the CD spectra for the absorbing side chains of W, Y, F, C, and M, and this correction can be substantial. With correction both W and F become good helix formers. The free energy for helix propagation was calculated using the Lifson-Roig statistical model for each of the nineteen amino acids at each point in their titration. The results show that the rank order of helical propensity for the nineteen amino acids changes with solvent environment. This result will be particularly important if proteins undergo hydrophobic collapse before secondary structures are formed, because amino acids can then see different solvent environments as the secondary structures are formed. Related amino acids are found to have interesting correlations in the shape of their titration curves. This finding provides one explanation for the limiting 70% accuracy in predicting secondary structure from sequence, since the helical propensities used are calculated for an average solvent environment. Proteins 2000;39:132-141.

Comparative evaluation of externally supported Dacron and polytetrafluoroethylene prosthetic bypasses for femorofemoral and axillofemoral arterial reconstructions. Veterans Affairs Cooperative Study #141.

PURPOSE: Currently, the choice of a vascular prosthesis for an extra-anatomic arterial bypass graft is left to the surgeon's preference because well-designed comparative evaluations have not been performed. The Department of Veterans Affairs Cooperative Study 141 was organized to identify whether there is improved patency with different prosthetic grafts for patients with femorofemoral or axillofemoral bypass grafts. METHODS: Between June 1983 and June 1988, patients at 20 Veterans Affairs Medical Centers who had aortoiliac occlusive disease but were not considered suitable candidates for aortic bypass surgery were randomized to receive either an externally supported polytetrafluoroethylene or Dacron bypass graft for an extra anatomic bypass. Doppler-derived ankle brachial indices (ABIs) were determined before the operation and serially after the operation. Patients were seen in follow-up every 3 months for the first year and every 6 months thereafter. All patients were instructed to take 650 mg of aspirin each day for the duration of the study. A bypass graft was considered to be patent if the Doppler-derived postoperative ABI remained significantly improved (0.15 units above the preoperative value), and additional clinical information (such as subsequent ABIs, angiograms, or operations) did not contradict these observations. RESULTS: Three hundred forty patients with femorofemoral bypass grafts and 79 patients with axillofemoral or axillofemorofemoral bypass grafts were randomized. The indication for the bypass operation was limb salvage in 72% of the patients. The assisted primary patency rate for a Dacron bypass grafting was 79% at 1 year, 63% at 3 years, and 50% at 5 years; for polytetrafluoroethylene bypass grafting, the patency was 77% at 1 year, 62% at 3 years, and 47% at 5 years. CONCLUSION: The overall results of this prospective randomized study suggest that the current choices of prosthetic bypass grafting have similar long-term patency in patients who undergo femorofemoral or axillofemoral vascular reconstruction.

Bovine lens crystallins do contain helical structure: a circular dichroism study.

In order to settle a recent discussion on the secondary structure of lens crystallins, we have measured the circular dichroism (CD) spectra of alpha-, beta(H)-, and beta(L)-crystallin from 178 to 250 nm and of gamma-crystallin from 168 to 250 nm. The results were analysed by means of a newly developed algorithm that almost doubles the reliability of secondary structure prediction and that allows discrimination between alpha- and 3(10)-helical, and between extended and polyproline beta-type structure. The results indicate that the crystallins studied contain a non-negligible amount of alpha-helical structure, although at least 50% of it is in the form of single and/or distorted loops. In alpha-crystallin, which is related to the chaperones, the helical content is lower than in beta- and gamma-crystallin. In some cases, the helices may play a role in DNA binding by the crystallins.

Analyzing protein circular dichroism spectra for accurate secondary structures.

We have developed an algorithm to analyze the circular dichroism of proteins for secondary structure. Its hallmark is tremendous flexibility in creating the basis set, and it also combines the ideas of many previous workers. We also present a new basis set containing the CD spectra of 22 proteins with secondary structures from high quality X-ray diffraction data. High flexibility is obtained by doing the analysis with a variable selection basis set of only eight proteins. Many variable selection basis sets fail to give a good analysis, but good analyses can be selected without any a priori knowledge by using the following criteria: (1) the sum of secondary structures should be close to 1.0, (2) no fraction of secondary structure should be less than -0.03, (3) the reconstructed CD spectrum should fit the original CD spectrum with only a small error, and (4) the fraction of alpha-helix should be similar to that obtained using all the proteins in the basis set. This algorithm gives a root mean square error for the predicted secondary structure for the proteins in the basis set of 3.3% for alpha-helix, 2.6% for 3(10)-helix, 4.2% for beta-strand, 4.2% for beta-turn, 2.7% for poly(L-proline) II type 3(1)-helix, and 5.1% for other structures when compared with the X-ray structure.