A complete understanding of their function and regulation will therefore be critical to disrupt one of the most pathological effects of Plasmodium infections. In an effort

to improve functional annotation and increase our understanding of the parasite’s biology, a number of research groups have been leveraging biochemical metabolic profiling and metabolomics strategies (40). Metabolomics is the study of the entire repertoire of metabolites, i.e. small molecules such as amino acids, sugars and fatty acids that are known to perform critical functions in various biological processes. Correlation analyses of transcriptomics, proteomics and metabolomics data are a powerful way to identify new metabolic pathways as well as genes that encode for specific enzymatic functions (41,42). While the study of metabolomics in Plasmodium is still in its infancy, it has already uncovered important biological insights with possible implications in terms of adaptation, evolution and host–pathogen Deforolimus interactions (43–45). Functional genomics suffers from the lack of tools to analyse the malaria parasite’s genome. For example, gene silencing using RNAi cannot be used in Plasmodium because the machinery does not exist in the parasite; gene knockout experiments are time-consuming processes not Target Selective Inhibitor Library compatible with large-scale high-throughput analyses. However, in the past few years, a transposon-based mutagenesis approach in Plasmodium has been developed (46). A Plasmodium-specific

selection cassette was added to the lepidopteran transposon piggyBac and transfected in parasites together with a transposase-containing helper plasmid (47). Random insertional mutants are obtained by multiple integrations of the transposon at TTAA recognition sites. Recent studies used piggyBac-based approaches to validate candidate parasite-specific

secreted proteins (48) or identify genes that are essential for the parasite’s proliferation (49). Used in combination with other genomics and proteomics analyses, piggyBac-based strategies could provide a better understanding of the parasite’s biology and its interactions Gemcitabine cost with its hosts. The data of large-scale and functional genomic analyses must be accessible and intelligible for practical and efficient usage. The task belongs to the informatics and bioinformatics fields that can provide the necessary tools. Up to now, data depositary banks and the Web-based databases such as PlasmoDB (http://plasmodb.org/plasmo/) have greatly facilitated the access, the comprehensive visualization and the analysis of large data sets. Gene predictions and annotations, new drug target identifications and discoveries of vaccine candidates all resulted from various genome-wide analyses. However, it is critical that such resources remain well maintained and free for maximized accessibility. Indeed, a systemic view of the malaria parasite’s biology can only be achieved with the successful integration and accessibility of the data from various origins.

Most vaccine strategies have focussed on the larval stage of the hookworms; however, there is some evidence that resistance to later stages is possible (60). In repeated experimental hookworm infections, it could be seen that although the majority of the newly infected larvae migrated from the skin to the gut, only a small number could attach successfully to the gut wall (60). The total number of worms attached (previously patent plus new arrivals) seemed dependent on levels of eosinophilic inflammation of the gut wall, and so it appears that resistance to the later gut feeding stages of the parasite is possible. Interestingly, in human enteric infection with dog hookworm in an Australian

community (see later), much more pronounced inflammation was seen than that with human hookworm (61). SAHA HDAC research buy High levels of eosinophil infiltration in the gut wall caused inflammation and pathology. This inflammatory allergic response has been cited as

the cause of dog hookworm ejection from humans, and its absence in human hookworm infection (and dog hookworm infection in dogs) argues for active and species-specific suppression of the anti-hookworm response (62). Thus, eosinophilic attack of adult worms in the gut may lead to ejection of the parasite, but at the cost Omipalisib mw of inducing a destructive eosinophilic enteritis. Other vaccine strategies to attack the adult parasite are being developed, which may not cause damaging inflammation. One approach is to target the gut of the adult worm to prevent

it from successfully feeding. Hookworms ingest blood from ruptured capillaries in the host gut wall, where the blood is digested in the hookworm’s own gut and absorbed. A cascade of proteolytic enzymes carries out the digestion of host blood, and these enzymes can be considered ‘cryptic’ antigens – they are never exposed to the host immune system, and so an immune response is never raised against them. During the course of feeding, however, Bumetanide the hookworm gut is exposed to antibodies in the host blood, a phenomenon of which we are targeting in our vaccine development strategy (63). A vaccine candidate, aspartic protease-1 (APR-1), has been identified from the adult blood-feeding stage of the parasite; a vaccine targeting APR-1 is aimed primarily at preventing effective nutrient uptake in the gut of the adult hookworm, effectively starving it to death (64). APR-1 is a protease involved in the haemoglobin digestion cascade within the gut of hookworms (65). It has been shown to be effective against both A. caninum infection in dogs (64,66) and N. americanus in hamsters (67). Indeed, the proposed mechanism by which APR-1 vaccines protect the host is via the induction of antibodies that neutralize the enzymatic activity of the protease, thus rendering it unable to digest haemoglobin and other blood proteins (Figure 1).

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 selleckchem 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 see more 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 Nintedanib (BIBF 1120) 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.

To prevent chronic inflammation, the liver must modulate innate and adaptive immune responses to these diverse antigens [1, 3]. Conversely, the liver is an important organ in host defence against parasitic and microbial infections [4]. Thus, immune responses can be initiated in the liver to eliminate microbial infection [5-7]. Further understanding of the mechanisms Daporinad cost that determine the balance between immunity to pathogens and tolerance to diverse dietary and other antigens will provide new insights into the design of therapeutic strategies to regulate immunity in liver infection,

autoimmunity and transplantation. Hepatic B cells comprise approximately 5% of intrahepatic lymphocytes [8-10]. Limited studies have addressed the function

of hepatic B cells in vitro [11] and in the regulation of experimental autoimmune biliary disease [12-14]. It has been shown that LPS-treated hepatic B cells enhance the production of interferon (IFN)-γ by liver natural killer (NK)1·1+ cells [11] and promote liver inflammation in the non-obese diabetic (NOD).c3c4 mouse model of autoimmune cholangitis Cabozantinib price [13], suggesting that hepatic B cells can regulate hepatic immune responses positively. In contrast, the Toll-like receptor (TLR) ligands LPS (TLR-4) and cytosine–phosphate–guanosine (CpG) (TLR-9) can stimulate interleukin (IL)-10-producing regulatory B cells (Breg) (B10) and regulate immune responses negatively [15-17]. Given that LPS is delivered continuously by the liver via the portal blood, we hypothesize that the ability of

hepatic B cells to regulate immune responses positively might be due to a lack of LPS-activated Breg in the liver. In this study we demonstrate that, unlike splenic B cells, hepatic B cells lack B10 cells and comprise significantly smaller proportions of B1a and marginal zone (MZ)-like B cells [16]. In addition, when compared with liver conventional myeloid (m)DCs from B cell-deficient mice, those from B cell-competent wild-type mice were more immunostimulatory, as evidenced by higher levels of maturation marker expression Selleck Temsirolimus in response to in-vivo LPS stimulation, and by a greater production of proinflammatory cytokines following ex-vivo LPS stimulation. Male C57BL/6 (B6; H2b) and B6·129S2-Ighmtm1Cgn/J (μMT) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). B6·129P2-IL-10tm1Cgn mice (IL-10 reporter) were kindly provided by Dr David Rothstein (University of Pittsburgh). They were housed under specific pathogen-free conditions at the University of Pittsburgh School of Medicine, with unlimited access to food and water.

4c). Interestingly, Cox-2-deficient mice had an approximately

25-fold lower Blimp-1 protein expression compared with wild-type controls (Fig. 4c). This further demonstrates that B-cell differentiation is Cox-2-dependent. To determine if the reduced generation of CD38+ antibody-secreting cells was a result of impaired differentiation of human B cells, we investigated whether the expression of plasma cell transcriptional regulators was influenced. We assessed both mRNA steady-state levels and protein expression of Blimp-1 and Xbp-1, which are essential transcription factors necessary for plasma cell differentiation. Pax5, a transcription factor important for initiating and maintaining the B-cell phenotype, was also investigated. Purified human B cells from three different donors activated for 24, 48, 72 or 96 hr were treated with either DMSO (vehicle) or the Cox-2 selective inhibitor SC-58125. RNA was extracted mTOR inhibitor at each time-point, reverse transcribed,

and subjected to real-time PCR analysis for Blimp-1, Xbp-1 and Pax5 expression. Messenger RNA steady-state levels of each transcription buy Rapamycin factor were normalized to 7S control mRNA steady-state levels. Comparing levels of Blimp-1, Xbp-1 and Pax5 with freshly isolated B-cell mRNA demonstrated that Pax5 mRNA steady-state levels decreased following stimulation with CpG plus anti-IgM, while Blimp-1 and Xbp-1 expression was enhanced (Fig. 5a). The mRNA fold-expression decrease after Cox-2 inhibitor treatment was determined by dividing the normalized mRNA expression values of the vehicle-treated cells by the normalized values of the SC-58125-treated cells (Fig. 5b,c). Following treatment of three different human donors with SC-58125, Blimp-1 mRNA expression was decreased 2·6 ± 0·8-fold by 24 hr, Docetaxel in vivo 2·8 ± 1·2-fold by 72 hr and 3·3 ± 1·1-fold by 96 hr (Fig. 5b). At the 20-μm dose Blimp-1 levels were reduced by 3·6 ± 0·5-fold after 72 hr of incubation (Fig. 5c). Over the time–course,

Xbp-1 mRNA expression was decreased (1·9 ± 0·1-fold) in the presence of SC-58125 at 72 hr (Fig. 5b). By 96 hr after Cox-2 inhibitor treatment we observed a 2·9 ± 1·2-fold decrease. Treatment of B cells with 20 μm SC-58125 for 72 hr resulted in a 4·9 ± 0·6-fold decrease in Xbp-1 mRNA expression (Fig. 5c). In contrast, Pax5 mRNA expression was relatively unchanged following inhibition of Cox-2 (Fig. 5b,c). These new data indicate that inhibition of Cox-2 reduced mRNA transcript levels of the transcription factors, Blimp-1 and Xbp-1, which are essential for the differentiation of B cells to plasma cells. To further demonstrate that the decrease in Blimp-1 and Xbp-1 mRNA was seen at the translational level, protein was extracted from activated human B cells treated with vehicle or SC-58125. A Western blot containing these samples from two different donors was probed for the expression of Blimp-1, Xbp-1, Pax5 and GAPDH as a loading control (Fig. 5d).

In particular, a threshold for the minimal area Am of macrophages on the red layer (split point 3 in Fig. 1) Y-27632 solubility dmso and thresholds for the minimal areas As and Acs of single spores and clustered spores, respectively, were used on the green layer (split points 4–6 in Fig. 1). We used different thresholds for single spores and clustered spores, Acs < As, because largely overlapping fluorescence signals in the images appear for spores that are lying close together in clusters. Furthermore, to distinguish spores from artifacts in the images, thresholds for object roundness and object asymmetry were used in addition to the area feature (split

points 5 and 6 in Fig. 1). Here, object roundness was evaluated by approximating the ROI by an outer and inner ellipse and Raf inhibitor by computing the difference σ between the major axis of the outer ellipse and the minor axis of the inner

ellipse.[16] In contrast, the object asymmetry was computed from the ratio of the main axes rmax and rmin of an ellipse that was fitted to the ROI as α = 1 − rmin/rmax. Here, we distinguished again between thresholds for the roundness of single spores σss and clustered spores σcs, and similar for the asymmetry of single spores with threshold αss. We modified the implemented algorithm[16] to deal with the current image data by dividing the segmentation into two sub-steps. Here, we first computed for each image an intensity threshold automatically and then applied the multi-threshold segmentation algorithm. With regard to the size of the spores (see split points 4–6 in Fig. 1), we enforced only a lower but not

an upper threshold and by that enhanced the probability of detecting all spores to ensure that the number of missed spores was minimal, i.e. we were opting for a high recall. However, since this segmentation sub-step did not distinguish Acyl CoA dehydrogenase between ROIs that are single spores or clustered spores, a second segmentation sub-step was required where clusters of spores were split into single spores based on the features roundness and asymmetry. The ruleset distinguishes between phagocytosed and non-phagocytosed spores being adherent and non-adherent to macrophages (split point 7 and 8 in Fig. 1). The decision of the class memberships for spores was made on the blue layer, because due to the staining only adherent and non-adherent spores that were not phagocytosed appear in blue. ROIs are classified as spores or artifacts in the images depending on their average intensity I relative to the threshold value Is in the range of integer values between 0 and 255. We optimised the value of Is (see Table 1) by a validation procedure involving a manual classification on selected images. Finally, non-phagocytosed spores were classified as adherent or non-adherent to macrophages (split point 8 in Fig. 1) depending on whether or not they share a border with macrophages on the red layer.

1). A landmark study of 32 065 haemodialysis patients, mean follow-up of 2.2 years, reported that deaths from cardiac arrests were most common after the long 2

day inter-dialytic break (after long inter-dialytic break, 1.3 vs 1.0 deaths per 100 person-years on other days, P = 0.004).[42] The DOPPS investigators reported similar findings in haemodialysis patients from the United States, Europe and Japan.[43] Possible explanations are manifold, including hypervolaemia, circulatory collapse, or electrolyte and metabolite build-up between dialysis sessions. Potassium is important for regulation of trans-membrane potential of cardiac myocytes, and there is evidence to support the hypothesis that potassium shifts, relative hypokalaemia post-dialysis[44] and pre-dialytic find more hypokalaemia predispose to arrhythmia. In one multivariate

Cox regression analysis of the risk factors for SCD in 476 chronic haemodialysis patients, PI3K inhibitor pre-dialytic hyperkalaemia conferred 2.7-fold increase (95% CI = 1.3–5.9).[45] In an observational study of 81 013 haemodialysis patients, the optimum pre-dialysis serum potassium in respect of long-term survival was between 4.6 and 5.3 mmol/L.[46] In a review of 400 dialysis unit cardiac arrests, patients who were dialysed against a low potassium dialysate (0 or 1.0 mmol/L) were twice as likely to have had a cardiac arrest.[47] It has also been reported that a dialysate potassium of <2 mmol/L (or <3 mmol/L, if pre-dialysis potassium is <5 mmol/L) confers increased risk of SCD.[3, 6] Electrical conduction is also dependent on intra-cardiac calcium handling; a low calcium dialysate (1.25 mmol/L) is associated PIK3C2G with aberrations in cardiac conduction

as assessed by electrocardiography, such as increased QTc dispersion or prolonged QT interval.[48] In view of these findings, there is a need for future studies to concentrate on the composition of dialysate in the hope of reducing arrhythmia burden. High rates of fluid removal may result in intra-dialytic hypotension, myocardial stunning and injury. In turn, this may predispose to arrhythmia or circulatory collapse. In DOPPS, a large ultrafiltration volume (>5.7% of post-dialysis weight) conferred an HR of 1.15 for sudden death (defined as deaths due to arrhythmia, cardiac arrest and/or hyperkalaemia).[6] Similarly, in a case-control study of 502 haemodialysis patients who had a sudden cardiac arrest with 1632 age- and dialysis-vintage-matched controls who did not, increased ultrafiltration volumes conferred an adjusted OR of 1.11 (95% CI = 1.02–1.033, P = 0.02). A recent observational study reported that depressed heart rate variability is associated with fluid overload in chronic haemodialysis patients.[49] This may be one of the pathophysiological mechanisms by which fluid overload predisposes to arrhythmias.

Strips were rinsed briefly with 25% 1.5 M pH 8.0 Tris before SDS–PAGE was performed using Criterion 12.5% Tris-HCl Precast gels (Bio-Rad), run at 200 V for approximately 45 min. For each sample, two gels were run simultaneously, one for silver staining and another for

immunoblotting. Gels for silver staining were fixed individually in 0.1 L fixing solution [50% (v/v) methanol, 10% (v/v) acetic acid] for selleck a minimum of 1 h, and were subsequently stained using a sensitive ammoniacal silver method based on silver nitrate. Gel images were acquired using the UMAX Powerlook 1000 flat-bed scanner. Proteins from unstained gels were transferred electrophoretically onto polyvinylidene fluoride (PVDF) membranes using the Trans-blot cell transfer system (Bio-Rad Laboratories). see more To visualize total proteins, membranes were stained with a Sypro Ruby blot stain (Bio-Rad

Laboratories). To detect immunoreactive proteins, membranes were destained and subsequently probed according to the Immun-Star™ WesternC™ kit protocol (Bio-Rad Laboratories). Membranes were immunolabeled with patients’ sera at a 1 : 250 dilution, and goat anti-human IgG antibodies coupled to HRP (1 : 2000; Bio-Rad) were used as a secondary antibody. The immunoreactive protein spots matched using both the Sypro Ruby stained membrane and the silver-stained gels were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Briefly, spots were washed twice for 10 min in 200 μL of 100 mM NH4HCO3, reduced at 37 °C for 1 h with 50 μL of 10 mM DTT, alkylated for 1 h in 50 μL of 10 mM iodoacetamide, washed for 10 min with 200 μL of 10 mM NH4HCO3, dehydrated in acetonitrile, and trypsin-digested with 10 ng μL−1 of trypsin (Promega, Annandale, NSW, Australia). After digestion for 14 h at 37 °C, peptides were extracted by washing the gel slice for 15 min with 25 μL 1% formic acid, followed by dehydration in acetonitrile. Digests were then dried in vacuo, resuspended in 10 μL 1% formic acid and submitted for

a Quadrupole-TOF analysis on a Micromass instrument which generated collision-induced dissociation. Results were analyzed using the Mascot MS/MS ion search (Matrix Science, Boston, MA), and searches were performed on the National Centre for Biotechnology OSBPL9 Information non-redundant (NCBI nr) database (specifically against the available genome sequence of C. concisus BAA-1457). This study was approved by the Research Ethics Committees of the University of New South Wales and the South East Sydney Area Health Service-Eastern Section, Sydney (Ethics No.: 03/163, 03/165 and 06/164). Recently, an association between the presence of C. concisus DNA and newly diagnosed CD was reported in two case–control studies using intestinal biopsies and fecal samples (Zhang et al., 2009; Man et al., 2010c). In addition, significantly higher levels of C. concisus-specific IgG antibodies were detected in children with CD as compared with controls (Zhang et al.

Conditioned media from cells were assayed for the levels of IL-8 and TNF by sandwich ELISA [DuoSet kit (R&D Systems)] according to the manufacturer’s instructions. This work was supported by Science Foundation

Ireland and Enterprise Ireland. Professor Paul Moynagh is a Science Foundation Ireland Principal Investigator (SFI 07/IN.1/B972). Gemma Kinsella is an Irish Research Council for Science, Engineering and Technology (IRCSET) postdoctoral fellow. The authors acknowledge the SFI/HEA Irish Centre for High-End Computing (ICHEC) and the HEA Trinity Centre for High Performance Computing (TCHPC) for the provision of computational facilities and support. The authors acknowledge the support of Openeye Scientific, Scitegic and Chemical Computing Group. Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal Alpelisib manufacturer disease by altering enterocyte physiology and producing mucosal inflammation. Many details concerning the host response against EPEC remain unknown. We evaluated the role of EPEC virulence factors on the inflammatory

response through an analysis of bacterial recognition, cell signalling, and cytokine production using an in vitro epithelial cell infection model. Interestingly, we found that EPEC infection recruits Toll-like receptor 5 (TLR5) to the cell surface. We confirmed that type 3 secretion system (T3SS) and flagellin (FliC) are necessary for efficient extracellular Selleck Erlotinib regulated kinases 1 and 2 (ERK1/2) activation and found that intimin could down-regulate this pathway. Besides flagellin, intimin dipyridamole was required to keep nuclear factor kappa B (NF-κB) activated during infection. EPEC infection activated tumour necrosis factor alpha (TNF-α) production and induced interleukin (IL)-1β and IL-8 release. Virulence factors such as intimin, T3SS, EspA and fliC were required for IL-1β secretion, whereas intimin and T3SS participated in IL-8 release. Flagellin was essential for late secretion of TNF-α and IL-8 and intimin stimulated cytokine secretion. Initial adherence limited TNF-α release, whereas late attachment

sustained TNF-α secretion. We conclude that intimin modulates TLR5 activation and intimate adherence alters the proinflammatory response. Enteropathogenic Escherichia coli (EPEC) causes paediatric diarrhoea worldwide [1]. EPEC infects enterocytes and produces elimination of the microvilli and actin-rich pedestal-like structures upon where bacteria adhere. This histological lesion is called ‘attachment and effacement’ (AE) [2]. The AE lesion results from a pathogenic process that comprises cell signalling transduction and bacterial intimate adherence [3]. EPEC contacts the cell in the initial adherence through adhesins and bacterial appendages, including the flagellum [4]. The bacteria establish a type 3 secretion system (T3SS), a complex structure that constitutes a ‘molecular syringe’ [5].

Parasite persistence and concomitant immunity were achieved by Lm/CpG 10, 11 in the absence of lesions. In order to understand and exploit the immunological features of Lm/CpG, we have continued to unravel how the immune response Lumacaftor cost to this vaccine is different from natural infection (leishmanization). We have discovered that Lm/CpG promotes the early proliferation of dermal Th17 cells, contrasting with the highly polarized Th1 response that takes place much later in mice vaccinated

with L. major alone. Most importantly, Th17 cells appear to be the predominant effector population in Lm/CpG-vaccinated mice, although Th1 cells are also present. Neutralization of IL-17 (confirmed by the use of IL-17 receptor-deficient mice) causes enhanced susceptibility to L. major infection (higher parasite burdens, development of lesions), accompanied by a decrease in IFN-γ production, in neutrophil migration, and by an increase in Treg frequencies. The intradermal model of infection produces an immunologically “silent”

phase during the first 2–3 wk 13, 14. We have reported that the combination of live parasites and CpG DNA eliminates such a phase by causing a Adriamycin order rapid activation of DC, release of proinflammatory cytokines, and migration of activated lymphocytes to the vaccine site 10, 11. We obtained a full cytokine profile of the vaccination site of mice immunized with the live vaccines (L. major or leishmanization versus Lm/CpG) or with CpG DNA alone as a control. We extracted cells from the dermis of vaccinated animals prior to vaccination (wk 1), and 2 wk (“silent” phase for L. major, activated phase for Lm/CpG), 6 wk (acute phase for L. major), and 10 wk post vaccination (chronic phase). Cells were restimulated ex vivo with the vaccines to determine the production of various cytokines in the culture supernatants. As shown in Table 1, we found significant differences in the time frame of the immune response among the experimental groups. Cytokines Baf-A1 in vitro were secreted at low levels in the uninfected skin (wk 1). As reported by us 10, 11, IL-6 production was significantly increased during the “silent” phase (wk 2)

in Lm/CpG-vaccinated mice. IL-12, TNF-α, IL-17, and IFN-γ were elevated at the same time point, confirming the early proinflammatory response initiated by Lm/CpG vaccination. TGF-β secretion was slightly elevated in the Lm/CpG when compared with L. major alone, although it was very low. Conversely, IL-10 secretion was lower in the ears of the Lm/CpG-vaccinated mice at this time point; again, the overall values were close to the limit of detection. Although IL- 4 secretion was higher in the Lm/CpG-vaccinated animals at wk 2, its level was very low at all time points and all groups, as expected from the genetically resistant C57BL/6 mouse. Wk 6 values revealed a reversal in cytokine profiles, with the L. major-vaccinated animals now showing a proinflammatory response significantly dominated by the production of IL-12 and IFN-γ.