4 We performed preliminary data analysis on anemia management and outcomes in 1,276 patients undergoing hemodialysis (HD) and enrolled in the CRC for ESRD. The patients were enrolled between July 2009 and June 2011 and were followed until December

2011. The mean age of patients undergoing HD was 59.6 years. Of the entire cohort of patients, 58.4% were male, 52.4% had a history of diabetes, and 43.3% (n = 552) were incident patients. At enrollment, the mean hemoglobin (Hb) level of the entire cohort, the incident patients, and the prevalent patients were 9.9 ± 1.7 g/dL, 8.8 ± 1.7 g/dL, and 10.7 ± 1.2 g/dL, respectively. ESAs were prescribed in 76.4% of the entire cohort, with a median dose of 8,000 units/week of epoetin in 70.9% of incident patients and 80.9% of prevalent patients. Intravenous iron was prescribed selleck chemical in 8.1% of the entire cohort, 9.2% of the incident patients, and 7.3% of the prevalent patients. The mean levels of TSAT and serum ferritin were 30.6% ± 15.9% and 292.9 ± 307.6 ng/mL, respectively. Hb levels correlated positively with serum albumin levels and dialysis adequacy

(Kt/V), whereas it correlated negatively with serum ferritin and high-sensitivity C-reactive protein (hs-CRP) levels. Multivariate linear regression analysis identified serum albumin (β = 0.408; P < 0.001) and Kt/V (β = 0.129; P < 0.001) and serum hs-CRP (β = -0.070; P = 0.006) as independent predictors Selleckchem EMD 1214063 for anemia. Sixty incident patients (10.8%) and 77 prevalent patients (10.6%) died

during the mean follow-up of 19.4 ± 8.5 months. The most common cause of death was infectious disease. After adjusting for age, dialysis vintage, comorbidities, iron status, and ESA dose, a lower Hb level was associated with mortality in the entire cohort. With an Hb level of 10–11 g/dL as a reference, hazard ratios associated with time-dependent Hb levels were 5.12 (2.62–10.02) for Hb levels <9.0 g/dL and 2.03 (1.16–3.69) for Hb levels 9–10 g/dL. In summary, compared with the international practice pattern for anemia management, intravenous iron administration was much lower in patients enrolled in CRC 5-FU clinical trial for ESRD. In addition, the survival benefit of higher Hb (>11.0 g/dL) levels was not seen in this Korean observational cohort. 1. KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease. Kidney Int. 2012; 2(4): 1–64. 2. Pisoni RL, Bragg-Gresham JL, Young EW, Akizawa T, Asano Y, Locatelli F, Bommer J, Cruz JM, Kerr PG, Mendelssohn DC, Held PJ, Port FK. Anemia management and outcomes from 12 countries in the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis. 2004; 44(1):94–111. 3. Fuller DS, Pisoni RL, Bieber BA, Port FK, Robinson BM. The DOPPS practice monitor for U.S. dialysis care: update on trends in anemia management 2 years into the bundle. Am J Kidney Dis.

9±5.5 (P=0.003). To determine whether the advantage of B. parapertussis was manifest at earlier stages of infection, mice (four per group) were inoculated as in the previous experiment with a mixed 1 : 1 inoculum of B. pertussis and B. parapertussis and euthanized at days 1, 2, 4 and 7 postinoculation. Doxorubicin research buy The competitive advantage of B. parapertussis was observed as early as 24 h postinoculation (mean CI=7) and was maintained through the peak of infection (Fig. 1b). Together, these data indicate that B. parapertussis not only outcompetes B. pertussis in a mixed infection, but also that it benefits

from the presence of B. pertussis in the infection. To further explore the competition between these two organisms in a mixed infection, mice (four per group) were infected with mixed inocula Pirfenidone in vitro at ratios (B. pertussis to B. parapertussis) of 10 : 1, 3 : 1, 1 : 3 and 1 : 10 (106 total CFU). Mice were euthanized 7 days postinoculation, and the bacterial load and ratio of the two organisms were determined as before. Bordetella parapertussis outcompeted B. pertussis

at all inoculum ratios (Fig. 2). In mice where the inoculum contained B. parapertussis as the predominant strain (1 : 3 and 1 : 10), B. pertussis was at a significant disadvantage, with relatively low CFU recovered from the mice (no B. pertussis was recovered from two mice in the 1 : 10 group). Remarkably, even when the inoculum contained 10-fold excess of B. pertussis (10 : 1), greater CFU of B. parapertussis were recovered from the host, with a mean CI of 31 (Fig. 2). Overall, these data show that B. parapertussis is able to outcompete B. pertussis in a mixed infection over a range of input ratios and apparently gains a greater advantage (higher CI) when the initial inoculum contains higher numbers of B. pertussis. To determine

whether the advantage to B. parapertussis occurs early in the process of infection, experiments were conducted staggering the inoculation with the two organisms. Two different staggered inoculations were tested: (1) mice were inoculated first with 2.5 × 105 CFU B. pertussis (t0) and were inoculated new 3 h later with 2.5 × 105 CFU B. parapertussis (t3), or (2) mice were inoculated initially with 2.5 × 105 CFU B. pertussis (d0) and inoculated 24 h later with 2.5 × 105 CFU B. parapertussis (d1). Mice from each group were euthanized on day 2 post-B. pertussis inoculation, and the bacterial loads and the ratio of the two organisms recovered were determined. In mice inoculated at t0 and t3, greater numbers of B. parapertussis than B. pertussis were recovered (CI=1.82, P<0.05, data not shown). When the inoculations were staggered by 24 h, neither strain had a significant advantage (CI=0.9, data not shown), even though B. pertussis colonization began 24 h earlier than that of B. parapertussis. These data indicate that B. parapertussis can outcompete B. pertussis during early infection even when B.

However, as shown in Fig. 5B, the intensity and position of the bands of ODN1668 at incubation time 0 were not affected by the check details change in the ratio of DNase I-treated ODN1720 to ODN1668. These results

suggest that the DNase I-treated DNA does not bind to ODN1668. Therefore, other mechanism than the nucleotide binding to ODN would be involved in the DNase I-treated DNA-mediated increase. Therefore, other mechanisms than these should be involved in the increased cytokine production by DNase I-treated DNA. In recent reports, the conformational changes of both TLR9 and CpG DNA were shown to be an important process for the activation of the TLR9 pathway. CpG DNA allosterically changes the TLR9 protein to

the dimer accessible to CpG motif and MyD88, which results in the activation of NF-κB and cytokine release 30. In addition, TLR9 recognition requires an intramolecular or intermolecular double-stranded DNA region at the position of the CpG motif and single-stranded DNA region at the 5′ end 31, 32. Conformational changes in TLR9 would not be involved in the DNase I-treated ODN1720, because the TNF-α production induced by A-type or B-type CpG ODN, other TLR9 ligands, was not increased by DNase I-treated DNA. These ligand-dependent effects of DNase I-treated ODN1720 could be explained by assuming that DNase I-treated ODN1720 has some direct effects on ODN1668 and pCMV-Luc, both of which are the only two PO DNA used in the present study. One possible ACP-196 molecular weight mechanism

is that DNase I-treated DNA alters the conformations of PO-CpG ODN into forms with a high ability to interact with TLR9 protein. This hypothesis is also compatible with the results of an absence of significant effects of DNase I-treated ODN on the non-CpG lipoplex-induced TNF-α production (Fig. 2A), which was mediated by receptors other than TLR9 18, 19. see more Further studies are needed to identify the mechanism for the increase in the cytokine release by DNase I-treated DNA. It is reported that DNase I-deficient mice and humans have anti-DNA antibody with high frequency and are prone to SLE 33, 34. Moreover, the DNase I activity was lower in SLE patients than in the control group 35. In the sera of DNase I-deficient individuals, an increasing amount of undegraded self-DNA containing CpG motifs can be an exacerbating factor of CpG-dependent immune response. For the purpose of treatment for lupus nephritis, in which the deposition of self-DNA/anti-DNA antibody complex in glomeruli is thought to be crucial for the disease pathogenesis, recombinant human DNase I was intravenously administered into the patients. Although serum hydrolytic activity of recombinant human DNase I was achieved after administration, there were no significant changes in serum inflammatory cytokines, including TNF-α and IL-6 36.

Together, these data suggest a novel mechanism of immunosuppression by dexamethasone. To induce immune synapse formation, untransformed resting human peripheral blood (PB) T cells were incubated with superantigen (Staphylococcus aureus enterotoxin B, SEB) loaded APCs. The immune synapse formation was analyzed using multispectral imaging flow cytometry (MIFC), which combines fluorescence click here microscopy and flow cytometry. MIFC allows the spatial quantification of fluorescence signals within T cells by defining regions of interest for the measurement (Supporting Information Fig. 1). T-cell/APC couples were identified by gating on cell clusters

according to DNA content (Hoechst33342 staining) and CD3 expression (Fig. 1A, blue gate). T-cell/T-cell couples (Fig. 1A, green gate) or cell clusters that contained more than one T cell or APC (Fig 1A, black gate) were eliminated from further analysis. Then, the accumulation of the TCR/CD3 complex and LFA-1 in the T-cell/APC contact zone was used as measure for immune synapse formation. As expected, in the absence of superantigen most T cells did not show an enrichment of TCR/CD3 and LFA-1

in the contact zone (Fig. 1B, left side). DAPT in vivo In the presence of SEB, however, T cells showed a clear formation of an immune synapse (Fig. 1B, right part). To quantify the number of T cells with an immune synapse, we acquired up to 25 000 T cells. Figure 1C shows the frequency of primary human T cells that showed an enrichment of TCR/CD3 and LFA-1 in the contact zone from 19 different donors. The mean number of

T cells with an immune synapse increased significantly in the presence of SEB. It is important to note that the variations of T cells forming immune synapses were relatively high between different donors, ranging from 0.2 to 1.5% (Fig. 1C). We therefore compared the values from experiments that were performed in triplicates to evaluate the variance in dependent samples (Fig. 1D). The mean standard deviation of the triplicates Histamine H2 receptor (intratest SD) was 7.5 per 10 000 T cells. Taken the high variations between different donors (Fig. 1C) and the low variations of the triplicates (Fig. 1D) into account, we decided to normalize the following experiments by setting the numbers of T cells of one individual with synapses in the absence of dexamethasone as 1. To analyze the effects of glucocorticoids on the formation of an immune synapse in untransformed human T cells, PB T cells were preincubated with the glucocorticoid dexamethasone (5 μM). This concentration inhibited blast formation and cell-cycle entry without having any toxic effects (Supporting Information Fig. 2). Interestingly, in dexamethasone pretreated T cells, an inhibition of TCR/CD3 and LFA-1 accumulation and thus immune synapse formation could be observed (Fig. 2A and B). The reduced maturation of the immune synapse was due to a combined failure of LFA-1 (Fig. 2C) and CD3 (Fig.

From our previous study (Pokkali et al., 2009), an MOI of 3 was found optimum for infecting PMNs, and hence, same was kept as standard throughout this study. Because we aimed at observing the initial effect of mycobacterial vaccine strains on neutrophils, early time point

of 4 h was chosen. Uninfected neutrophils (Control) served as negative find more control, and 10 nm phorbol myristate acetate (PMA) (Sigma Chemicals)–stimulated cells were used as positive control. After 4 h, the neutrophil culture supernatants (Nu sups) were collected, centrifuged, and used to stimulate peripheral blood mononuclear cells (PBMCs), and the remaining was stored in aliquots at −70 °C until use. The cells were washed with PBS twice and used for fluorescence-activated cell sorting (FACS) staining protocol as given in the section ‘cell phenotyping click here by flow cytometry’. The buffy coat containing PBMCs was collected after Ficoll-Hypaque density gradient centrifugation. The cells were washed once with Hanks’ balanced salt solution (HBSS) and suspended in RPMI 1640 medium supplemented with 1% FBS. The cell viability was always found to be > 95% through trypan

blue exclusion test, and the cell density was adjusted to 1 × 106 mL−1. The cells were stimulated with 200 μL of infected Nu sups and cultured in 12 Well Clear TC-Treated Multiple Well Plates (Corning

Life Sciences) for 18 h at 37 °C in a humidified 5% CO2 incubator. After 18 h, the cells were harvested and stained for FACS as given in the section ‘cell phenotyping by flow cytometry’. Cell Tangeritin surface expression of CD32, CD64, TLR-4, and CXCR3 on neutrophils (CD16+ve); CD69 and CXCR3 on T helper cells (CD4+ve); and CCR5 and CCR7 on monocytes (CD14+ve) was determined by staining the cells using the monoclonal mouse anti-human conjugated antibodies, i.e. CD16 (clone 3G8)–fluorescein isothiocyanate (FITC), TLR-4 (clone HTA125)–phycoerythrin (PE), CD32 (clone FL18.26), CD64 (clone 10.1), CD4 (clone RPA T4), CD14 (clone M5E2)–allophycocyanin (APC), CD69 (clone FN50)–phycoerythrin-cyanine5 (PE-Cy5) (BD Pharmingen), and CCR5 (clone 45549)–FITC, CCR7 (clone 150503), CXCR3 (clone 49801)–PE (R & D Systems), and their fluorescence emission was detected in FL-1 (FITC), FL-2 (PE), FL-3 (PE-Cy5), and FL-4 (APC) channels. The above specified clones were used throughout the study. Briefly, cells were incubated with PBS containing the combinations of antibodies at saturation for 20 min at 4 °C. Cells were washed and fixed with 1% paraformaldehyde (Sigma Chemicals) in PBS and analyzed on a FACSCalibur flow cytometer (Becton Dickinson).

IV inoculated parasites reach the selleck kinase inhibitor liver within minutes (26), whereas sporozoites inoculated into the skin slowly trickle out of the inoculation site over a period of 1–3 h (27). Our results indicate that the lower parasite liver load after ID inoculation is unlikely to be explained by a delayed arrival

of sporozoites in the liver. Comparison of the parasite liver load at 35 h post-ID injection was still ±15 times lower compared to the parasite liver load at 30 h post-IV injection (Figure 2). Despite differences between parasites species, including among others infectivity (28) or host cell preference (29–31), our data in P. berghei parallel previous results in P. yoelii studies (25). Therefore, the relatively low level of parasites capable of reaching the liver after ID injection is likely a common feature among Plasmodium species.

CD8+ T cell responses are known to be essential for protection induced by attenuated live sporozoite immunization in rodent models. Our data corroborate previous studies on P. berghei RAS-induced immunity showing expansion of CD8+ memory T cells, mainly in the liver, together with high IFNγ production in IV immunized selleck chemical mice (12–15). The low immune responses observed after ID immunization likely follow the low parasite liver load. RAS ID and subcutaneous immunization of human volunteers also show low protection levels, and in nonhuman primates and mice subcutaneous or ID immunization lead to lower GBA3 IFNγ responses compared to IV sporozoite immunization

(18). Despite the differences in phenotyping and gating strategy, CD8+ effector (memory) T cells (CD44hi CD62L-) and not central memory T cells (CD44hi CD62L+) are identified as induced T-cell subset. In another study using the P. yoelii model, major CD8+ T cell responses were generated in the draining lymph nodes after infected mosquito bites or ID inoculation of sporozoites. Although parasite liver load was reduced, complete protection defined as impediment of blood-stage infection was not evaluated (32). We did not test the regional lymph nodes response and cannot exclude a possible contribution but our data clearly demonstrate that ID inoculation is inefficient in inducing protection. In addition, a measure of sporozoite load in regional lymph nodes following ID inoculation would have been informative. Unfortunately, in vivo visualization of PbGFP-Luccon is not possible because of a relatively low luciferase expression at the sporozoite stage (22). Next to cellular components, antibody responses can contribute to protection by whole sporozoite immunization (8). Our data suggest that induced functional antibodies may contribute to protection but are more likely related to exposure.

This of course was one of the key points noted by Tolman (1932) and demonstrated decades later by Harlow (1959). That is, the so-called secondary reinforcers Trametinib mw (e.g., curiosity, contact comfort) were incorrectly characterized as derived from primary reinforcers rather than having primary status on their own. Problem 2 was the fact that the natural environment is filled with high levels of ambiguity—that is, given the myriad of events that co-occur, it is unclear whether a stimulus is causally related to another stimulus

(or to a reward) or whether these co-occurrences are merely coincidences that lead to suspicious attributions of causal relations. How does the naïve (infant) learner resolve this ambiguity without the benefit of top-down knowledge that is only available to a mature learner? The road to addressing these two problems was paved by a second wave of methodological advances in the study of infant learning in the 1970s and 1980s and then a third wave of interest in what has become known as statistical learning in the 1990s and 2000s. A key methodological advance was the development and elaboration of the habituation paradigm by Bornstein (1985), Fantz MAPK Inhibitor Library (1964), Horowitz (1974) and McCall and Kagan (1970). They showed that repeated exposure

to a stimulus led to a decline in a criterion response (e.g., looking

time), which could then be reactivated by a change in that stimulus. Although this simple habituation paradigm provided an excellent measure of discrimination, it was the addition of a “family” of stimuli during the so-called multiple-habituation phase that allowed the paradigm to address questions of category learning. In the hands of Cohen and Strauss (1979) and Fagan (1976), the multiple-habituation paradigm allowed investigators to ask how infants grouped stimuli into categories without the involvement of any conditioned response or primary reinforcer—infants looked for the Avelestat (AZD9668) sake of looking and learned for the sake of learning. Paradigms that followed in the tradition of operant conditioning, using motor responses other than looking time such as sucking or foot-kicking, showed that infants as young as 1 day after birth were excellent learners. Siqueland and De Lucia (1969) demonstrated that infants suck to turn on a stimulus. Rovee-Collier, Sullivan, Enright, Lucas, and Fagan (1980) demonstrated that infants kick to wiggle a stimulus, despite the absence of any other reinforcer. And DeCasper and Fifer (1980) showed that newborns suck differently (by starting or delaying a burst of sucks) to one class of auditory stimuli over another.

2E). To further confirm these findings, we purified CD4+

T cells from B6 BCG-vaccinated and unvaccinated DLNs at different time points postvaccination and measured cytokine mRNA induction in these cells. Consistent with data shown in Fig. 2D, IL-17 mRNA induction occurred in CD4+ T cells earlier than the induction of IFN-γ mRNA, which was detected on day 14 postvaccination (Fig. 2F). Together, our data show that BCG vaccination induces an early IL-23-dependent Th17-cell response that precedes the Th1-cell response, and is required for the induction of an effective BCG vaccine-induced Inhibitor Library chemical structure Th1-cell response. Th17 cells are induced early in vivo following BCG vaccination and are important for subsequent generation of vaccine-induced Th1 cells at later time points (Figs. 1 and 2). Therefore, we then addressed whether the Th1- and Th17-cell polarizing cytokines namely IL-12 or IL-23 are induced in DCs in response to BCG exposure. We found that following BCG exposure, DCs produced both IL-23 and IL-12 cytokines (Fig. 3A and B). Interestingly, BCG also induced high levels of the anti-inflammatory cytokine, IL-10 in BCG-exposed DCs (Fig. 3C). IL-10 is an anti-inflammatory cytokine that inhibits IL-12 production and Th1-cell differentiation 26. Accordingly, www.selleckchem.com/products/BIBW2992.html IL-10 also inhibits IL-12 production in BCG-infected

DCs and the generation of IFN-γ-producing cells 27. Based on these data, we hypothesized that the absence of early Th1-cell responses in vivo following BCG vaccination was due to high BCG-induced IL-10 levels (Fig.

3C) and that IL-17 dependence to induce Th1-cell responses (Fig. 1) was a host strategy to overcome the IL-10-mediated inhibition. To address this hypothesis, we first treated BCG-stimulated DCs with IL-10-neutralizing antibody and measured IL-12 production in supernatants. Mirabegron As expected 27, neutralization of BCG-induced IL-10 resulted in significantly increased production of IL-12 (Fig. 3D). We also determined the effect of IL-10 neutralization on Th1 cell generation by coculturing naïve OT-II TCR Tg T cells with BCG/OVA323–339-treated DCs in the presence of IL-10-neutralizing antibody. Consistent with our hypothesis, we report that T-cell-derived IFN-γ production was inhibited in the presence of BCG and neutralization of IL-10 reversed BCG-mediated inhibition of IFN-γ production in T-cell supernatants (Fig. 3E). These data suggest that despite induction of some IL-12 in BCG-exposed DCs, coincident induction of IL-10 inhibits Th1-cell responses. Importantly, Ag85B-specific Th1-cell responses detected in vivo were also increased in BCG-vaccinated il10−/− mice when compared with B6 BCG-vaccinated mice (Fig. 3F).

When administered intravenously UF heparin generally has a half-life approximating 1.5 h. UF heparin is highly negatively charged and binds non-specifically to endothelium, platelets, circulating proteins, macrophages and plastic surfaces. In addition to removal by adherence, Rucaparib datasheet heparin is cleared by both renal and hepatic mechanisms and is metabolized by endothelium. Interestingly, UF heparin has both pro- and anti-coagulant effects. Heparin can be directly procoagulant through platelet activation and aggregation. However, its main effect is anticoagulant,

through its binding to anti-thrombin (anti-thrombin III or heparin-binding factor I). At high doses heparin can also bind to heparin-binding factor II – which can directly inhibit thrombin. When heparin binds anti-thrombin it causes a conformation change, which results in a 1000–40 000× increase in the natural anticoagulant effect of anti-thrombin. Heparin-bound anti-thrombin inactivates multiple coagulation factors including covalent binding of thrombin and Xa and lesser inhibition of VII, IXa, XIa, XIIa. By inactivating thrombin, UF heparin inhibits thrombin-induced platelet activation as well. Of note, UF heparin-bound anti-thrombin inactivates thrombin (IIA) and Xa equally.

Only UF heparin with more than 18 repeating saccharide CX 5461 units inhibits both thrombin and Xa, whereas shorter chains only inhibit Xa. For haemodialysis, UF heparin can be administered, usually into the arterial limb, according to various regimens, but most commonly is administered as a loading dose bolus followed by either an infusion or repeat bolus at 2–3 h.9 The initial bolus is important to overcome the high level of non-specific binding, following which there is a more linear dose : response relationship. The loading dose bolus may be 500 units or 1000

units and infusion may vary from 500 units hourly to 1000 units hourly, depending on whether the prescription is ‘low dose heparin’ or ‘normal heparin’. Heparin administration usually ceases at least 1 h before the end of dialysis. The most important risk of UF heparin is the HIT syndrome (HIT Type II). Other risks or effects attributed to UF heparin that have been reported include why hair loss, skin necrosis, osteoporosis, tendency for hyperkalaemia, changes to lipids, a degree of immunosuppression, vascular smooth muscle cell proliferation and intimal hyperplasia.10–12 Beef-derived heparin can be a risk for the transmission of the prion causing Jacob Creutzfeld type encephalopathy.13 Depolymerized fractions of heparin can be obtained by chemical or enzymatic treatment of UF heparin. These are also anionic glycosaminoglycans but have a lower molecular weight of 2–9 kDa, mostly around 5 kDa – thus consisting of 15 or fewer saccharide units.

Each group was boosted with EG95 protein at 4 weeks post-immunization. Antibody levels were determined in 2-weekly collections of serum, Ganetespib by ELISA. The results are presented in Figure 3. All sheep infected with VV399 showed evidence of seroconversion. Animals primed with EG95 showed higher levels of priming with EG95 protein compared with animals immunized with VV399. However, there was no difference in the serological response of both groups of sheep to the booster injection of EG95 protein. Poxviruses are proven delivery vehicles for a wide range of antigens against various diseases (11–13). The secreted EG95 protein, produced during the oncosphere and post-oncospheral life stage

prior to metacestode formation, affords protection against hydatid disease in the intermediate host, and in this study, we explored the use of VACV as a delivery system for this antigen. Mice are a well-established species for testing the immune

response against foreign antigens expressed by recombinant VACV (11,19) and were used in this study to examine the antibody response against EG95 expressed from VACV and to examine the ability of the mouse antiserum produced to kill E. granulosus oncospheres in vitro. Whilst all groups infected with VV399 showed little antibody response to EG95 at 2 weeks post-infection, it was observed that the antibody response continued to increase with time, and by 42 days post-infection, antibody levels were comparable with mice Dasatinib immunized intraperitoneally with EG95 protein at 2 weeks post-immunization. Furthermore, it was clear that all animals were primed with VV399 as significant responses were detected in all animals either boosted with VV399 or EG95 protein. The primary antibody response observed was consistent with the immune response seen in foxes and skunks fed orally a sponge bait containing recombinant VACV expressing the rabies virus glycoprotein (20–22). In their experiments, neutralizing antibody Casein kinase 1 increased up to day 28–30, but decreased thereafter. In addition, the priming effect of VV399 was also observed by

the antibody levels produced in animals boosted intraperitoneally with EG95 protein (Figure 1). We found that a second immunization with VV399 enhanced the antibody response generated from previous exposure, albeit not to the same level as that seen with EG95 protein combined with alum adjuvant. The priming and boosting effect of EG95/HIS with alum is interesting. Previous work (16) has shown that in sheep, alum is a poor adjuvant compared with QuilA. It was necessary to use alum because QuilA was shown to be lethal in our mice. The level of immunity generated by the double exposure to VV399 intranasally prompts the suggestion that this recombinant virus could be used for immunizing grazing animals in the field.