Given the exciting immunotherapeutic potential of manipulating Treg-cell function in the context of infectious disease, autoimmune disorders, cancer and allotransplantation,96,97 studies of these cells in the dog have never been more timely. O.A.G. gratefully Sunitinib clinical trial acknowledges funding in his laboratory for work on canine regulatory T cells from the Biotechnology and Biological Sciences Research Council and Novartis Animal Health. We thank Dr John E. Peel for insightful discussions during the course of this work, Dr Iain Peters and

Mr Daniel Lowther for practical tips on RT-qPCR, Drs Ayad Eddaoudi and Philip Hexley for help with FACS™, and Professors Julian Dyson and Dirk Werling for help with tritiated thymidine assays. The authors have no conflicts of interest to disclose. “
“Expression features of genetic landscape which predispose an individual to the type 1 diabetes are poorly understood. We addressed this question by comparing gene expression profile of freshly isolated peripheral blood mononuclear cells isolated from either patients with type 1 diabetes (T1D), or their first-degree relatives or healthy controls. Our aim was to establish whether a distinct type of ‘prodiabetogenic’ gene expression pattern in the group Sorafenib clinical trial of relatives of patients with

T1D could be identified. Whole-genome expression profile of nine patients with T1D, their ten first-degree relatives and ten healthy controls was analysed using the human high-density expression microarray chip. Functional aspects of candidate genes were assessed using the MetaCore software. The highest

number of differentially expressed genes (547) was found between the autoantibody-negative healthy relatives and the healthy controls. Some of them represent genes critically involved in the regulation of innate immune responses such as TLR signalling and CCR3 signalling in eosinophiles, humoral Interleukin-3 receptor immune reactions such as BCR pathway, costimulation and cytokine responses mediated by CD137, CD40 and CD28 signalling and IL-1 proinflammatory pathway. Our data demonstrate that expression profile of healthy relatives of patients with T1D is clearly distinct from the pattern found in the healthy controls. That especially concerns differential activation status of genes and signalling pathways involved in proinflammatory processes and those of innate immunity and humoral reactivity. Thus, we posit that the study of the healthy relative’s gene expression pattern is instrumental for the identification of novel markers associated with the development of diabetes. Type 1 diabetes (T1D) is considered to be a T-helper 1 (Th1)-mediated disease characterized by an autoimmune destruction of the insulin–producing pancreatic beta cells [1, 2].

We found that CXCL2 effectively restored neutrophil infiltration into the inoculated corneas and caused typical CaK in nude mice (Fig. 7). In fact, coadministration

of CXCL2 with blastospores exacerbated the severity of CaK and neutrophil infiltration in the corneas of BALB/c mice (Fig. 7). We compared the effect of IL-17 neutralization in mice concurrently inoculated with Candida in ear skin and the cornea. Contrary to its effect in cornea, IL-17 neutralization worsened the infection in skin (Fig. 8A). Histological analysis revealed LY2157299 concentration that while IL-17 neutralization inhibited leukocytes infiltration at both sites, it led to fungal expansion in the skin (Fig. 8B and C). These results suggest that IL-17 inhibition elicits protective

and destructive responses in corneas and skin, respectively. The pathogenic role of lymphocytes in infectious keratitis has been previously reported in experimental models of other pathogens. Over three decades ago, it was noted that nude mice did not develop viral keratitis when challenged with the herpes PD-0332991 in vivo simplex virus [23]. Pearlman et al. showed that immunocompetent mice no longer developed Onchocerca volvulus keratitis when depleted of CD4+ cells [24]. By studying related mechanisms, Rouse and colleagues identified bystander activation of lymphocytes in the pathogenesis of herpes simplex keratitis [25, 26]. We report, for the first time, that CaK cannot be induced in either nude mice or CD4+ T-cell-depleted BALB/c mice, and that IL-17 is a critical factor in CaK initiation. We further showed that neutrophils and CD4+ T cells (supposed Th17 cells) are the main producers of IL-17

during CaK initiation (Fig. 4 and 5). On the other hand, Treg cells GABA Receptor and γδ T cells, which are key players in other systems [27, 28], were not involved in CaK formation in cornea (Supporting Information Fig. 2). Though the differential roles of these cell types in CaK and herpes simplex keratitis could be explained by the significant difference in the properties of the two pathogens, more extensive studies are needed to investigate why Treg cells and γδ T cells are not seemingly involved in pathogenesis of FK. Lastly, the differential effects of IL-17 neutralization on CaK and fungal dermatitis in the same mouse (Fig. 8) underscore the duality of IL-17 activity and the importance of cellular context in the pathogenesis of keratitis [29-33]. Thus, the effects of C. albicans may not be recapitulated by other fungal genera. While highlighting a critical role for IL-17 in CaK initiation, our results also bring to light several intriguing questions concerning corneal infections. The first involves the mechanism of efficient fungal clearance in corneas of nude mice. It has been proposed that structural features, as well as some innate factors, afford corneas the ability to hinder pathogens [34] or blastospore-pseudohypha transformation [35].

Methods:  Fifteen primary renal transplant centres (15/17; 88% response rate) and 21 secondary renal

transplant centres (21/24; 88% response rate) responded to an online survey addressing key questions investigating their current practice in the nutritional management BAY 73-4506 of adult KTR. Results:  Referral from primary to secondary sites was limited with only two sites (9%) routinely receiving referrals. Allocated funding for KTR at secondary sites was low (n = 4, 14%). Many primary sites received nil or <0.5 full-time equivalent (FTE) funding for inpatient (n = 8, 53%); and nil or ≤0.2 FTE funding for outpatient services (n = 9, 60%). In sites reporting FTE hours, the average dietitian-to-patient

ratio was 1 FTE dietitian for every 383 (range 50–1280) annually transplanted patients. Major barriers identified in delivering nutrition services at primary sites included time/lack of resources and limitations with systems to identify or track transplant recipients. Conclusion:  Dietitian-to-patient ratios in the management of KTR at primary sites are inconsistent and likely to be inadequate at secondary transplant sites to implement guideline recommendations, especially for weight management. Investigations into the effectiveness of innovative Ibrutinib price interventions such as groups or telehealth are warranted, which may assist practitioners to achieve guideline recommendations in an environment of limited resources. “
“Uraemia is characterized by intestinal bacterial Bcl-w translocation, which contributes to the development of microinflammation. Probiotics enhance the intestinal barrier and overall health of the host. The present study investigated whether the probiotic Bifidobacterium animalis subsp. lactis Bi-07 alleviates bacterial translocation and ameliorates microinflammation in experimental uraemia. Sixty Sprague–Dawley rats were divided into three groups of 20 rats each: the sham group, which underwent only laparotomy; the uraemia group, which underwent 5/6 nephrectomy; and the uraemia + probiotic group, which

underwent 5/6 nephrectomy and daily intragastric administration of B. animalis subsp. lactis Bi-07 for 4 weeks. Bacterial translocation was evaluated by polymerase chain reaction amplification of the green fluorescent protein (GFP) gene from oral GFP-labelled Escherichia coli in the peripheral blood, mesenteric lymph nodes, liver, and spleen. Intestinal permeability, plasma inflammatory biomarker levels, and endotoxin levels were measured. Jejunum, ileum, and colon specimens were removed for histological examination. Uraemic rats exhibited a significantly higher incidence of bacterial translocation (70%) than did sham rats (10%). Probiotic treatment resulted in a decrease in bacterial translocation (20%).

These results are in agreement with the observation that blocking IL-2 signaling impairs Th17 differentiation [29], which is disabled in Pim1TgγcKO cells. Collectively, here we documented that Pim1 permits survival and functional maturation of CD4+ T cells in the absence of γc, but that lineage differentiation in the periphery still required γc signals that could not be replaced by Pim1. To understand the role of γc signaling in T-lineage cells, here we aimed to reconstitute γc deficiency by overexpressing Pim1. Using Pim1TgγcKO mice, we specifically asked whether Pim1 would be

sufficient to replace γc requirement in T-cell development and survival. While Pim1 improved CD4+ αβ T-cell development CP-690550 solubility dmso and restored peripheral CD4+ T-cell numbers, it failed to do so for other T-lineage cells, including CD8+ T cells, CD4+ Treg cells, NKT cells, CD8αα IELs, and γδ T cells. Thus, in contrast to all other T-lineage cells, CD4+ T cells are unique to require γc signaling primarily for prosurvival purposes and to be γc independent in their lineage specification and differentiation. Classically, γc cytokines had been considered essential for T-cell development because of their prosurvival effects. γc signaling induces BGB324 expression of antiapoptotic

molecules such as Bcl-2 and Mcl-1 [12, 30], and it inhibits proapoptotic factors such as Bax, Bad, and Bim [31-33]. Accordingly, Bax deficiency significantly restored thymopoiesis in IL-7 receptor deficient mice, and Bcl-2 overexpression improved T-cell development

in γc-deficient mice [34-36]. However, antiapoptotic effects alone are insufficient to fully account for γc requirement in T-cell development. Also, the Bcl-2 effect on increased thymocyte numbers itself is conflicting, with studies arguing for improved differentiation versus mere increase of developmentally MYO10 arrested thymocyte numbers in Bcl-2 transgenic mice [16, 35-37]. Thus, the survival function of γc is presumably more complex than solely providing antiapoptotic signals. In this regard, recent studies showed that trophic effects of γc signaling are also critical components of its survival function. In fact, prometabolic activities were found to be important also for CD4+ T-cell differentiation [38, 39] and for determining CD8+ cytotoxic T-cell fate [40, 41]. Thus, prometabolic activity is another important arm of the γc cytokine signaling pathway. The Pim1 kinase epitomizes the full range of γc survival effects as it induces both antiapoptotic and prometabolic pathways. Pim1 inactivates Bad to prevent apoptosis, and it activates 4E-BP1 and S6 kinase to upregulate metabolism [19, 23, 42]. In resting T cells, Pim1 is expressed below detectable levels, but IL-7 stimulation in vitro potently induces Pim1 expression [19].

Whilst denosumab is not renally cleared, little is known about its effects and safety in patients with severe CKD. Methods: We performed a study of all patients with CKD stage IV or V administered denosumab since 1/1/2010 at Austin Health. Patients were identified by cross-referencing pharmacy administration records with patient’s renal function prior to drug administration. Data was collected and analysed retrospectively by chart review for clinical parameters, including calcium levels prior to and following administration PD-0332991 solubility dmso of denosumab. Results: 8 patients with stage V and 5 patients with stage IV CKD were identified. 6 of 8 patients with CKD V, and 2 of 5 patients with

CKD IV had significant hypocalcaemia, (corrected calcium < 2.0 mmol/L), with the lowest

corrected calcium being 1.18 mmol/L. Of these 8 patients, 3 patients had significant life-threatening complications requiring intensive monitoring. For patients who developed hypocalcaemia, the median time to serum calcium nadir was 26 days (range 10–56 days) and the median time to normalise calcium level was 86 days (range 15–140 days). Treatment of hypocalcaemia required large doses of calcium and vitamin D and increases to dialysate calcium, consistent with hungry bone syndrome. Conclusions: Patients with advanced CKD are at greatly increased risk of severe hypocalcaemia and hungry bone syndrome click here when administered denosumab. Denosumab is best avoided in patients with advanced CKD but if used very close monitoring is required. 174 RITUXIMAB-ASSOCIATED HYPOGAMMAGLOBULINAEMIA: INCIDENCE,

OUTCOMES AND EFFECT OF DOSE IN PATIENTS WITH MULTI-SYSTEM AUTOIMMUNE DISEASE DM ROBERTS1,2, RB JONES1, RM SMITH1, F ALBERICI1,3, DS KUMARATNE1, S BURNS1, DRW JAYNE1 1Addenbrooke’s Hospital, Cambridge, UK; 2University of Queensland, Brisbane, Australia; 3University of Parma, Italy Aim: To describe the incidence, severity and predictors of hypogammaglobulinaemia from rituximab for small vessel vasculitis and other multi-system autoimmune diseases, Phospholipase D1 and clinical outcomes following IgG replacement therapy. Background: Hypogammaglobulinaemia has occurred after rituximab treatment of lymphoma and rheumatoid arthritis but data are scarce for other autoimmune indications. Methods: Retrospective study in a tertiary referral specialist clinic. The severity of hypogammaglobulinaemia was categorised on the basis of the nadir serum IgG concentration measured during clinical care. Clinical details of patients prescribed IgG replacement therapy were reviewed. Results: 288 patients received rituximab; 243 were eligible for inclusion with median follow up of 42 months. 26% patients were IgG hypogammaglobulinaemic at the time rituximab was initiated and 56% had IgG hypogammaglobulinaemia during follow-up (5–6.9 g/L in 30%, 3–4.9 g/L in 22% and <3 g/L in 4%); IgM ≤ 0.3 g/L in 58%. The nadir IgG was non-sustained in 50% of cases with moderate or severe hypogammaglobulinaemia.

Various-sized fluorescein-labelled ISF tracers were stereotactically inoculated into the striatum of adult mice. At times from 5 min to 77 h, uninfected and scrapie-infected mice were compared. C57BL/10 mice expressing wild-type anchored PrP, which develop non-amyloid PrPres similar to humans with sporadic Creutzfeldt–Jakob disease, were Idasanutlin mw compared with Tg44+/+ mice (transgenic mice secreting anchorless PrP) expressing anchorless PrP, which develop amyloid PrPres similar to certain human familial prion diseases. In C57BL/10 mice, extensive non-amyloid PrPres aggregate deposition was not associated with abnormal clearance

kinetics of tracers. In contrast, scrapie-infected Tg44+/+ mice showed blockage of tracer clearance and colocalization of tracer with perivascular PrPres amyloid. As tracer localization and clearance was normal in infected C57BL/10 mice, ISF blockage was not an important pathogenic mechanism in this model. Therefore, ISF blockage is unlikely to be a problem in non-amyloid human prion diseases such as sporadic Creutzfeldt–Jakob disease. In contrast, partial ISF blockage appeared to be a possible pathogenic

mechanism in Tg44+/+ mice. Thus this mechanism might also influence human amyloid prion diseases where expression of anchorless or mutated PrP results in perivascular amyloid PrPres deposition and cerebral amyloid angiopathy. “
“F. P. Roche, B. J. Sheahan, S. M. O’Mara and G. J. Atkins (2010) Neuropathology and Applied Neurobiology36, 648–660 Semliki Forest virus-mediated gene therapy of the RG2 LY2109761 price rat glioma Aims: Glioblastoma multiforme is the most common and most malignant adult brain tumour. Despite numerous advances in cancer therapy there has been little change in the prognosis of glioblastoma multiforme, which remains invariably fatal. We examined the Semliki Forest virus virus-like particle (SFV VLP) expression system encoding interleukin-12 (IL-12) as a therapeutic intervention against the syngeneic

RG2 rat glioma model. Methods: Glioma-bearing rats were treated with IL-12-encoding SFV VLPs via an implanted cannula. Animals were treated with 5 × 107 (low-dose) or 5 × 108 Branched chain aminotransferase (high-dose) VLPs per treatment and the effect on glioma growth and survival was assessed. Results: Low-dose treatment produced a 70% reduction in tumour volume, associated with a significant extension (20.45%) in survival that was dependent upon IL-12 expression. High-dose treatment resulted in an 87% reduction in tumour volume, related to the oncolytic capacity of the SFV VLP system. VLP delivery to the central nervous system (CNS) demonstrated the potential of the vector system to induce lethal pathology that was unrelated to replication-competent virus or high-level IL-12 expression. Treatment-related death was pronounced in high dose-treated animals and appeared to be the result of inflammation, necrosis and oedema at the inoculation site.

Moreover, in the subgroup of MS-275 cost patients without previous immunosuppressant treatment, there was no disability progression during the treatment period. Hence, mycophenolate mofetil might serve as an alternative therapy for RRMS [41]. Moreover, recent studies examined the safety and efficacy of combinations of ‘classic’ immunosuppressive

drugs with recombinant IFN-β and showed equivocal results [42]. Moreover, some novel oral immunomodulatory drugs have recently been tested alone or in combination with IFN-β or GA in Phase III trials in patients with CIS or RRMS (see below). A parallel approach, however, is lacking in CIDP. Mitoxantrone is an anthracenedione derivative related to the anthracyclines doxorubicin and daunorubicin. It interacts with topoisomerase-2, stabilizes its cleavable complex with DNA, and thus prevents the ligation of DNA strands and consecutively delays cell-cycle progression. Preparations and administration: mitoxantrone is approved in Europe for the disease-modifying monotherapy of patients with highly active RRMS and SPMS

mTOR inhibitor (‘escalation therapy’) [43]. Its use, however, is limited by cardiotoxicity (the standard cumulative lifetime dose of mitoxantrone is 96 mg/m2, which can be extended up to a maximum lifetime dose of 140 mg/m2 under careful risk–benefit weighting and monitoring) and the risk of therapy-associated leukaemia (especially acute myelogenous leukaemia, AML). Given these limitations and the broadening spectrum of drugs available for patients with highly active RRMS, the use of mitoxantrone is limited in clinical practice to patients with SPMS. Mitoxantrone is administered intravenously at a dosage of 12 mg/m2 every 3 months for a total of 2 years, according to the mitoxantrone

in MS study (MIMS) [44]. To extend the total administration period, the dosage can be reduced to 5 mg/m2 upon clinical stabilization. Decitabine ic50 Clinical trials: there are no recent clinical trials with mitoxantrone in MS. Moreover, due to a lack of evidence from randomized, controlled clinical trials the use of mitoxantrone in CIDP is not established. Adverse effects, frequent: secondary amenorrhoea/azoospermia, nausea and vomiting, myelosuppression; infrequent: alopecia, cardiotoxicity, secondary leukaemia (especially AML) [45, 46]. Contraindications: severe active infections, chronic or relapsing infections, cardiomyopathy, treatment with other cardiotoxic drugs, severe liver or kidney dysfunction, pregnancy and lactation. Due to a lack of evidence from randomized, controlled clinical trials, the use of cyclophosphamide in MS and CIDP is not properly established [25, 47]. Teriflunomide is the biologically active metabolite of leflunomide, which is approved for the treatment of rheumatoid arthritis.

This choice was based on the knowledge that all members of the γc cytokine family signal through the IL-2Rγc (7). Ascending parasitemia following the i.p. injection of 1 × 106 parasitized erythrocytes was similar in both groups of mice, reaching peak values of 20.7 ± 12.5% on day 9 post-inoculation (PI) in knockout DAPT mouse (KO) mice lacking functional genes for the expression of the IL-2Rγc peptide and 11% on day 7 in control mice. Whereas parasitemia in control mice was suppressed to approximately 0.01% by day 13 PI, parasitemia in IL-2Rγc−/y mice remained at high unremitting levels (8–29%) for >7weeks PI when the experiment was terminated

(Figure 1a). This finding that parasitemia was prolonged at high levels in IL-2Rγc−/y mice indicates that signalling through the IL-2R complex is essential for the suppression of P. c. adami parasitemia. Acute blood-stage P. c. adami infections in mice

are suppressed by antibody-mediated immunity (AMI) dependent on CD4+ T cells and B cells (21) and/or cell-mediated immunity (CMI) dependent on CD4+ T cells and γδT cells (22,23). The observation that IL-2Rγc−/y selleck chemical mice failed to clear P. c. adami parasites from their blood indicates that both AMI and CMI against the parasites were defective in these mice lacking a functional IL-2R owing to a mutation of a single gene, the IL-2Rγc gene. IL-2Rγc−/y mice have been reported previously by others to be deficient in αβ T cells, γδT cells and B cells (3,4). As indicated in Table 1, we observed similar deficiencies in Mannose-binding protein-associated serine protease these cell populations. Because IL-2 and IL-15 may have redundant roles in immunity to blood-stage malaria, we determined the time courses of P. c. adami parasitemia in IL-2/15Rβ−/− mice and intact controls following inoculation with 1 × 106 parasitized erythrocytes.

Parasitemia was prolonged in IL-2/15Rβ−/− mice by approximately 3 weeks as compared to control mice (Figure 1b), but the mice eventually cured. Both γδ T cells and B cells were deficient in the spleens of IL-2/15Rβ−/− mice compared with infected control mice (Table 1) with numbers similar to those seen in IL-2Rγc−/y mice. In addition, antibodies reactive with crude malarial antigen were detected in the sera of IL-2/15Rβ−/− mice, following the suppression of parasitemia albeit at approximately half the concentrations seen in control mice (Table 2). This difference was not statistically different. Both IL-2 and IL-15 stimulate through the IL-2/15Rβ (9,13). Whereas IL-2-deficient mice exhibit P. c. adami parasitemia of prolonged duration before spontaneously clearing (11), the effects of IL-15 deficiency on the course of malaria caused by the adami subspecies of the parasite had not yet been determined. To assess whether IL-15 contributes to the suppression of acute parasitemia, we compared time courses of P. c. adami parasitemia initiated with 1 × 106 parasitized erythrocytes in IL-15 KO mice vs. C57BL/6 controls.

Also to note, most of the previous studies have been performed in male mice, known to be more territorial than female, particularly in super-enriched cages. In fact, that is the reason why females are generally the choice in animal models of chronic infection. In the

present study, we used cage enrichment based on present European recommendations [8]. This simple and inexpensive enrichment does not seem to induce stress, even in the groups housed ZD1839 with intermittent access to environmental enrichment, as indicated by thymic cellularity. Environmental enrichment has a long history in experimental psychology and neurobiology. Over the last 15 years, a razing interest in environmental enrichment as a way to adapt the cage to the animals was observed. Drawing on the 3Rs principle of animal experimentation (Replacement, Reduction and Refinement) [43], this approach may be described as refinement of animal housing. While the aim of environmental enrichment in psychology and neurobiology has been to create cage conditions that induce differences in a number of experimental behavioural parameters; the aim of the environmental

enrichment as housing refinement is to modify the housing conditions to improve animal welfare, with a minimum effect on behaviour and physiological parameters, and consequently, interfering as little as possible with experimental results. However, concern that altering the Pexidartinib nmr housing of laboratory rodents may influence the results of experiments [9, 10] is delaying the routine implementation of environmental enrichment as housing refinement.

The environmental enrichment design chosen for our experiment was based on preference and motivation tests showing that nesting material and shelter are resources that mice are motivated to access [3, 5–7]. We show that introducing such enrichment, and thus implementing the European recommendations for laboratory animal accommodation, does not compromise current animal models of chronic infection, and can be applied with no concern by researchers in the field. Conceived and designed the experiments: Anna Olsson and Margarida Correia-Neves; Performed the experiments: Andreia Costa, Claudia Nobrega and Susana Roque; Protein tyrosine phosphatase Analysed the data: Anna Olsson, Andreia Costa, Claudia Nobrega, Susana Roque and Margarida Correia-Neves; Wrote the paper: Anna Olsson and Margarida Correia-Neves. This work was supported by grants from the ECLAM and ESLAV Foundation. CN and SR are recipients of PhD fellowships from Portuguese Foundation for Science and Technology (FCT). “
“Secondary lymphoid organs function to increase the efficiency of interactions between rare, antigen-specific lymphocytes and antigen presenting cells, concentrating antigen and lymphocytes in a supportive environment that facilitates the initiation of an adaptive immune response.

GLA-SE was an efficient adjuvant for the generation of gag-specific CD4+ T-cell responses in spleen and lymph nodes (Fig. 1 A and B, respectively). We had previously shown that LPS and its analogue MPLA were weak adjuvants for inducing CD4+ T-cell responses to HIV gag p24 delivered within anti-DEC antibody when compared with poly IC as the adjuvant 4, 26. Similar results were obtained when

we used GLA-SE as an adjuvant and injected the protein vaccine s.c. (Supporting Information Fig. 1). To test if GLA-SE as an adjuvant could induce cell-mediated immune responses at a mucosal site, as is likely helpful to protect against certain diseases, we assessed the presence MAPK Inhibitor Library chemical structure of antigen-specific T cells

in the lungs and lamina propia of mice immunized by the s.c. route. Surprisingly, after injection of anti-DEC-HIV gag p24 or nontargeted gag-p24 protein along with GLA-SE, we could detect gag-specific CD4+ T cells in a magnitude similar to four times bigger than spleen and lymph nodes (Fig. 1C and D). To evaluate the type of cellular response induced by GLA-SE to a protein vaccine, we measured the production of Th1, Th2, and Th17 cytokines in supernatants of splenocytes stimulated with p24-peptides. In agreement with a previous publication using GLA-SE to adjuvant Fluzone vaccine 27, we found that gag-specific T cells induced by GLA-SE produced IFN-γ but not IL-17 or Th2 cytokines, verifying that GLA-SE allows a protein Progesterone vaccine to induce a polarized Th1 T-cell response (Fig. 1E). To determine if the new synthetic Selleck Dinaciclib TLR4 agonist GLA-SE could also generate a robust antibody response to protein vaccines, the sera of mice immunized with GLA-SE and anti-DEC-HIV gag p24 vaccine or nontargeted gag-p24 were assayed for anti-HIV gag antibody by ELISA. As expected from prior work with Fluzone, GLA-SE but not SE alone, adjuvanted strong antibody

responses (Fig. 1A). Specific IgG1, IgG2b, and IgG2c titers against p24 antigen were detected with the GLA-SE adjuvant but not with the control emulsion (Fig. 2B–D). It is known that LPS as well as its analogue, MPLA, are good adjuvants for antibody responses 4, 32, 33. Our results indicate that GLA-SE is also effective at inducing antibody responses. To prove that TLR4 was required in vivo, we assessed GLA-SE function in WT and TLR4−/− mice and found that similar to LPS, HIV-gag-specific T-cell and antibody responses were abolished in TLR4-deficient mice (Fig. 3A–C). Thus GLA-SE, a nontoxic derivative of LPS that is known to signal through TLR4 in vitro 34, 35, also requires TLR4 to act as an adjuvant in vivo. To begin to obtain evidence that DCs were required for the adjuvant action of GLA-SE, we compared the response of DEC-targeted HIV gag p24 with soluble HIV p24 protein. All concentrations of anti-DEC-HIV gag p24 tested, 0.5–5.