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In this study, B cell function in protective TH2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα−/− mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4+ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88−/− B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4+ T cell-mediated protective immunity against N. brasiliensis infection.
Author Summary
Parasitic nematode infections are an extremely important global public health problem. Infections by hookworms and roundworms for example cause anemia, widespread developmental problems and devalued immunity against bacterial infections such as salmonella and tuberculosis. Although treatable with drugs, parasitic nematode re-infections occur as humans do not develop protective immunity. Ultimately, the public health burden caused by these infections will be best controlled by the development of vaccines against nematode infections. For these to be effective, it is important to understand how the various components of the immune system can respond to infection. In this study, we show that B cells, which typically protect against infection by producing antibodies, can also protect against an experimental hookworm like nematode infection by additional mechanisms. This form of protection instead depended on B cells producing cytokines associated with parasitic nematode expulsion and also by providing T cells with specific instruction. Together, these B cell driven responses lead to a rapid resolution of the infection. These important findings indicate that vaccination strategies against nematode parasites such as hookworms need to understand immune responses other than antibody to be optimally protective.
Citation: Horsnell WGC, Darby MG, Hoving JC, Nieuwenhuizen N, McSorley HJ, Ndlovu H, et al. (2013) IL-4Rα-Associated Antigen Processing by B Cells Promotes Immunity in Nippostrongylus brasiliensis Infection. PLoS Pathog 9(10): e1003662. doi:10.1371/journal.ppat.1003662
Editor: William C. Gause, University of Medicine and Denistry New Jersey, United States of America
Received: December 13, 2012; Accepted: August 4, 2013; Published: October 24, 2013
Copyright: © 2013 Horsnell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Research Foundation (NRF) South Africa, the Medical Research Council (MRC) South Africa and the South African Research Chair Initiative of the Department of Science and Technology (DST) to FB and the International Centre for Genetic Engineering and Biotechnology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Parasitic nematode infections are a significant global public health burden. Infections with Ascaris lumbricoides, Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus occur in a third of the world's population [1]. Individuals frequently suffer from repeated infections and do not develop robust immunity against re-infection [2]. Such infections are significant causes of morbidity, with hookworm infections, for example, being a major cause of childhood anemia in many endemic areas [3]. Effects on cognitive development, as a result of repeated childhood infections have been reported [4], and parasitic larval migrations through the host may exacerbate chronic lung pathologies in endemic areas [5], [6]. To date no licensed vaccines exist against these parasites. To accelerate their development a detailed understanding of host immunity is essential, especially extra intestinal immunity against infective stage larvae [7]. Studies in humans and experimental models of infection have established that TH2 immune responses drive host resolution of primary infections [8], [9].
Key to effective expulsion of murine model parasites, such as Nippostrongylus brasiliensis, Heligmosomoides polygyrus and Trichuris muris, is host expression of IL-4Rα [10]. IL-4Rα is an essential component of the heterodimeric receptors required for IL-4 and IL-13 signalling, which ultimately drive host immune polarisation to TH2. Use of IL-4Rα−/− mice has clearly demonstrated an absolute requirement for IL-4Rα expression in resolving primary nematode infections. This is dependent on IL-4Rα expression on non-hematopoietic cells [11] including smooth muscle cells [12] and epithelial cells [13], [14]. However, IL-4Rα expression on hematopoietic cells does impact on the magnitude of the hosts TH2 response to N. brasiliensis. For example, disruption of IL-4Rα expression on CD4+ T-cells results in a significantly reduced TH2 response to primary N. brasiliensis infection [15] and contributes to optimal control of secondary infection [16]. However, it is not known how IL-4Rα expression on other hematopoietic cells contributes to protection from N. brasiliensis re-infection.
Our understanding of cellular mechanisms underlying protective immunity to helminth re-infection has, until recently, been limited. Protective immunity to nematode infection can occur both in the intestine, in the case of primary N. brasiliensis infection and both primary and secondary H. polygyrus infections, while immunity to secondary N. brasiliensis infections occurs in the lung. In the case of the strictly intestinal parasitic nematode H. polygyrus, rapid resolution of re-infection requires alternatively activated macrophages [17], CD4+ T cells [18], parasite specific type 2 antibody responses [19], [20] and B cell cytokine production [21]. In human infections such as A. lumbricoides and hookworms, which have some analogy to N. brasiliensis infections, the parasites are not confined to the intestine. Here larval migrations through the circulatory and pulmonary systems have resulted in these sites playing important roles in infection induced pathology and parasite killing [7]. Studies with N. brasiliensis show host responses in the lung play a key role in the rapid resolution of re-infection [7], [22]. Furthermore, roles for eosinophils [23], basophils [24] and CD4+ T cells [16], [25], but not B cells [20], in coordinating this immunity have also been demonstrated.
The work we present here addresses how B cells in secondary lymphoid organs (lymph nodes and spleen) can rapidly contribute to the control of recall immunity to N. brasiliensis re-infection. We then test if transfer of these potentially protective B cells can confer protection in a naïve mouse against primary exposure to N. brasiliensis infection. Using this approach, we identified key roles for B cells in immunity to the parasite. Strikingly, this was associated with production of IL-13 by IL-4Rα-sufficient B cells and also required MHCII compatibility and MyD88-expression. Thus, B cells can rapidly contribute to immunity to pathogens through multiple effector functions.
IL-4Rα is essential for immunity against re-infection with N. brasiliensis
To identify a possible role for IL-4Rα in generating protective immunity IL-4Rα−/lox (IL-4Rα sufficient) and IL-4Rα−/− mice were infected with N. brasiliensis. Infection was subsequently cleared by drug treatment before re-infection with 500 L3 larva. Intestinal worm burden was quantified at different time points post-secondary infection (Figure 1A). IL-4Rα−/− mice had significantly higher intestinal worm burdens compared to IL-4Rα−/lox mice at day 5 or 7 post-secondary infection (Figure 1B). Loss of IL-4Rα was associated with multiple defects in known effectors of host TH2 immunity, including decreased mucus production in the lung (Supplementary Figure S1A), decreased IgG1 production and decreased IL-13 production by CD4 T cells and B cells in the lung draining lymph node (Figure 1C–E).
IL-4Rα−/− and IL-4Rα−/lox mice were infected for 5 or 7 days post-secondary N. brasiliensis infection (A). Intestinal worm burdens were then quantified (B). Pulmonary mucus production was established by PAS staining (C). Serum Antibody titers of N. brasiliensis specific IgG1 were determined by ELISA (D). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B-cell populations (E). IL-4−/−, IL-13−/− and IL-4Rα−/lox mice were infected for 5 days post-secondary N. brasiliensis infection and intestinal worm burdens were then quantified (F). Pulmonary mucus production was established by PAS staining (G). Serum Antibody titers of N. brasiliensis specific IgG1 were determined by ELISA (H). Data is representative of 3–4 independent experiments. n = 4–6 mice per group.
IL-4Rα dependent immunity to N. brasiliensis re-infection is driven by IL-13 signalling
To investigate whether resolution of secondary N. brasiliensis infection was dependent on IL-4 and/or IL-13 signalling via IL-4Rα re-infection studies were repeated in IL-4−/− and IL-13−/− mice. Here, the significantly higher intestinal worm burden at day 5 secondary infection in IL-13−/− mice compared to IL-4−/− mice (Figure 1F), demonstrated IL-13 signalling through IL-4Rα is essential for immunity against re-infection with N. brasiliensis. This higher worm burden could was also be associated with an absence in goblet cell mucus production in IL-13−/− mice, but not IL-4−/− mice (Figure 1G and Figure S1B). IL-13−/− mice demonstrated equivalent N. brasiliensis specific IgG1 antibody titers as IL-4Rα−/lox mice. Conversely, IL-4−/− mice demonstrated reduced specific IgG1 responses (Figure 1H). Taken together, these data (Figures 1) indicate that IL-4-depedent antigen specific IgG1 antibody responses may not be required for optimal immunity to re-infection, but that IL-4Rα-mediated IL-13 signalling is required.
IL-4Rα-responsive B cells producing IL-13 are required for effective immunity to N. brasiliensis re-infection
IL-4Rα-mediated effects on B cell function during N. brasiliensis re-infection were investigated in MB1CreIL-4Rα−/lox BALB/c mice, which have B cell-specific abrogation of IL-4Rα expression [26]. Secondary infection resulted in a significantly higher intestinal worm burden in MB1CreIL-4Rα−/lox mice when compared to IL-4Rα−/lox mice (Figure 2A). Whilst goblet cell hyperplasia in MB1CreIL-4Rα−/lox mice was equivalent to that seen in control IL-4Rα−/lox mice (Figure 2B and Figure S1C). Antigen specific IgG1 was significantly drastically reduced (Figure 2C). Interestingly, IL-13 cytokine production by T and B cells was also reduced in MB1CreIL-4Rα−/lox mice, when compared to IL-4Rα−/lox mice (Figure 2D and Figure S2). Together, these studies show that a loss of IL-4Rα on B cells is sufficient to impair immunity to N. brasiliensis re-infection.
MB1CreIL-4Rα−/lox and IL-4Rα−/lox mice were infected for 5 or 7 days post-secondary N. brasiliensis infection and intestinal worm burdens were then quantified (A). Pulmonary mucus production was established by PAS staining (B). Serum Antibody titers of N. brasiliensis IgG1 were determined by ELISA (C). Mediatstinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B cell populations (D). B cells were isolated from N. brasiliensis infected BALB/c, MBcreIL-4Rα−/lox and IL-13−/− and transferred into naïve BALB/c mice (E). Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were then established at day 5 post infection (F & G). The results shown represent 2–4 independent experiments. n = 4–7 mice per group.
To demonstrate if IL-4Rα responsive and IL-13 competent B cells can directly confer protection against primary N. brasiliensis infection, we adoptively transferred B cells isolated from infected IL-4Rα−/lox, IL-13−/− or MB1CreIL-4Rα−/lox mice into naïve BALB/c mice (Figure 2E and Figure S3). Transfer of antigen-experienced IL-4Rα-responsive B cells into naïve BALB/c mice (WT+WT B-cells) reduced intestinal worm burdens. In contrast, transfer of primed B cells deficient for either the IL-4Rα or IL-13 did not reduce intestinal worm burden (Figure 2F, G). These results support our previous observations (Figures 1 & 2A) that IL-4Rα-responsive and IL-13 competent B cells contribute to protective immunity against N. brasiliensis.
Previous studies have shown that in the absence of B cells control of N. brasiliensis re-infection is similar to WT mice [20]. Our experiments also found that the host's ability to control N. brasiliensis re-infection did not require B cells (Figure 3A). Nevertheless, this does not exclude the possibility that antigen-experienced B cells are able to modulate the response in normal mice. To test this experimentally, we transferred B cells from infected IL-4Rα−/lox or infected MB1CreIL-4Rα−/lox mice into naive μMT mice (Figure 3B). Transferred N. brasiliensis primed IL-4Rα responsive B cells augmented protection in μMT mice (Figure 3B), whereas transferred B cells from infected MB1CreIL-4Rα−/lox mice did not. This B cell IL-4Rα-dependent protection correlated with significant increases in MST CD4+ T cell IL-13 production (Figures 3C). Together these results show that the immune response can compensate for the absence of B cells, but the introduction of pathogen-experienced IL-4Rα-responsive B cells can accelerate protective immunity against N. brasiliensis.
N. brasiliensis infected μMT and BALB/c mice were re-infected with 500xL3 larvae and at day 5 post-secondary infection, the intestinal worm burdens was quantified (A). The possible role for IL-4Rα expressing B cells in boosting immunity independently of endogenous B cells was determined by transfer of B cells isolated from N. brasiliensis infected IL-4Rα−/lox (WT B cells) or MB1Cre IL-4Rα−/lox (IL-4Rα−/− B cells) into naïve μMT mice. These mice were then infected with 500xL3 N. brasiliensis and worm burdens quantified at day 5 post infection (B). Mediastinal lymph node CD3+CD4+ T cell populations IL-13 responses (C) were established by FACS staining. Results shown represent 2 independent experiments. n = 4–7 mice per group.
Enhanced B cell ability to interact with T helper cells is required for optimal immunity to N. brasiliensis re-infection
Optimal host control of N. brasiliensis re-infection was associated with B cell IL-13 production and enhanced CD4+ T cell TH2 responses. A further way B cells could contribute to T cell responses is through cognate, physical interactions that are associated with antigen-presentation through MHCII and co-stimulatory molecule expression. As a first step in demonstrating, if such an interaction is also a feature of IL-4Rα-dependent immunity to N. brasiliensis re-infection, we initially assessed expression of CD86 and MHCII on CD19+ B cells from naive and N. brasiliensis re-infected mice. CD86 and MHCII expression was equivalent between naive mice of both groups (Figure 4A, lower panel). In re-infected mice, CD86 and MHCII surface expression in MB1CreIL-4Rα−/lox mice was reduced when compared with IL-4Rα−/lox mice (Figure 4B, lower panel). CD4+ T cells showed no differences between the mouse strains in expression of CD28 and TCR. (Figure 4A and B, upper panel). Thus, IL-4Rα expression in B cells can help enhance the expression of B cell markers of activation after N. brasiliensis infection.
Surface expression of CD28 and TCR on CD4+ T cells and CD86 and MHCII on B cells in naive (A) and N. brasiliensis re-infected (B) IL-4Rα−/lox mice and MB1CreIL-4Rα−/lox mice was established by FACS analysis. Histograms: filled gray: isotype control, thin line: IL-4Rα−/lox, thick black line: MB1CreIL-4Rα−/lox. Contributions by MHCII dependent antigen presentation were demonstrated by isolating WT or MHCII−/− B cells from naive or infected mice then adoptively transferring into naive C57BL/6 mice (C). Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were established at day 5 post infection (D). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B cell populations (E). MHC dependent antigen presentation was confirmed by isolating WT and BALB/b B cells from naive or infected mice adoptively transferring into naive BALB/c mice. Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were established at day 5 post infection (F). Mediastinal lymph node IL-13 responses were established by intracellular FACS staining in CD4+ T-cell and B220+ B cell populations (G). Data is representative of 2 independent experiments. n = 4–6 mice per group.
These findings indicated B cell cognate interactions with T cells and also antigen presentation may contribute to optimal immunity against N. brasiliensis re-infection. Our data presented in Figures 1 and 2 also indicates that B cell immunity may be independent of antibody class switching. This may exclude involvement of highly specific clonally expanded populations of B cells. We therefore hypothesized that protection may instead be mediated by a rapidly modified B cell antigen presenting response to N. brasiliensis infection. To demonstrate the possible role for antigen presentation, we adoptively transferred MHCII−/− B cells from 1 day N. brasiliensis infected mice (Figure 4C) into naive mice. At the next day, mice were infected and days 5 post infection, recipients of MHCII−/− B cells showed significantly higher worm burdens than mice, which received control WT B cells (Figure 4D). Protection was associated with increased IL-13 production by both B and T cell populations in the mediastinal lymph node (Figure 4E). To further control MHCII dependency, similar infection experiments were carried out in BALB/b mice, which are unable to present antigen via MHCII to BALB/c B cells. BALB/c recipients of adoptively transferred N. brasiliensis-experienced BALB/b B cells showed also significantly higher worm burdens, when compared to mice which received BALB/c B cells from N. brasiliensis infected mice (Figure 4F). These results further support that MHCII-dependent antigen presentation by B cells does contribute to host immunity to N. brasiliensis. Again, protection was associated with increased IL-13 production by both B and T cell populations in the mediastinal lymph node (Figure 4G). Together, these results suggest that MHCII expression contributes to the B cell protective response to N. brasiliensis re-infection.
B cells can rapidly launch protective antigen-dependent responses to N. brasiliensis infection
Our results presented in Figure 4 suggested that antigen-experienced B cells can rapidly contribute to protection. We therefore tested in vivo whether immunity induced early in infection with N. brasiliensis is dependent on IL-4Rα-responsive B cells. Adoptive transfer of IL-4Rα responsive B cells from IL-4Rα−/loxp mice isolated from mice 1 day post N. brasiliensis infection into wild type mice (Figure 4C) [27], [28] enhanced protection (Figure 5A), but not adoptive transfer of IL-4Rα unresponsive B cells from MB1creIL-4Rα−/loxp mice. Furthermore, only transfer of IL-4Rα responsive B cells enhanced B and CD4+ T cell IL-13 responses in the lung (Figure 5B, C) and mediastinal lymph node (Figure 5D), strengthen the necessity of IL-4Rα-responsive B cells for protective immunity.
MBcreIL-4Rα−/lox and IL-4Rα−/lox mice were infected for 1 day with N. brasiliensis before spleen B cells were isolated and transferred into naive wild type mice (As in Figure 4C). These were infected with 500xL3 N. brasiliensis and intestinal worm burdens were quantified at day 5 post infection (A). Lung CD3+CD4+ and CD3+CD4+CD44hi T cell populations were analysed by FACS staining (B). Lung CD4+ T cell (C) and mediastinal lymph node CD4+ T cell and B220+ B cell population (D) IL-13 responses were established by intracellular FACS staining. Data is representative of 2 independent experiments. n = 4–6 mice per group.
This ability of B cells to confer protection so rapidly after parasite exposure further supports this response being independent of BCR. Other mechanisms of more rapid and possibly less stringent/polyfunctional antigen recognition by B cells may therefore play a role. Initial analysis does not support these transferred B cells conferring protection via an early production of IL-13 (Figure S5). However, rapid antigen processing and presentation may be mediated by B cells directly loading soluble peptide onto MHCII [29], [30] or via antigen internalisation and processing by Toll like receptors (TLR) [31], [32], [33].
To assess if rapid TLR mediated antigen processing contributed to reduced worm burdens, we repeated transfer experiments using B cells isolated from MyD88−/− mice at one day post infection. Mice, which received MyD88−/− B cells displayed significantly higher worm burdens than those which received B cells from WT 1 day infected recipients (Figure 6A). Protection was associated with increased IL-13 production by T cell populations in the mediastinal lymph node (Figure 6B). We then examined whether these effects were due to direct exposure of antigen by B cells by pulsing naive WT or MyD88−/− B cells overnight with N. brasiliensis antigen (Figure 6C). Transfer of B cells from MyD88−/− mice into naive mice resulted in impaired control of infection (Figure 6D), associated with lower IL-13 production by T cells in the mediastinal lymph node (Figure 6E). To rule out non-specific effects, we also pulsed wild type B cells with a range of antigens. We found that only B cells pulsed with N. brasiliensis antigen conferred a reduction in host intestinal worm burdens. Recipients of B cells pulsed with LPS (a potential bacterial contaminant during N. brasiliensis infection), ovalbumin and soluble Leishmania major antigen did not show any reduction in intestinal worm burden when compared to wild type controls (Figure S6). This data indicated that the reduction in worm burden that we see in recipients of N. brasiliensis pulsed B cells is pathogen specific. Moreover, pulsing of MHCII−/− B cells with N. brasiliensis also resulted in impaired reduction in worm burdens (Figure 6F). Together these data suggest an association between rapid pathogen specific MyD88 dependent antigen processing and MHCII antigen presentation by B cells underlying the accelerated host immunity to N. brasiliensis infection.
B cells were isolated from WT or MyD88−/− mice 24 hours post N. brasiliensis infection and adoptively transferred into naive WT mice (As in Figure 4C). 24 hours later these mice were infected with 500xL3 N. brasiliensis and subsequently killed 5 days post infection and worm burdens were counted (A). The mediastinal lymph node CD4+ T-cell IL-13 response was established (B). B cells were isolated from naive C57/BL6, MyD88−/− or MHCII−/− mice and pulsed with N. brasiliensis antigen overnight. These were then washed and transferred into naive C57/BL6 mice 24 h prior to infection (C). D5 PI intestinal worm counts are shown (D & F). The mediastinal lymph node CD4+ T-cell IL-13 response was established (E). Data is representative of 2 experiments, n = 5–7 mice per group.
This study demonstrated that IL-4Rα responsive B cells co-ordinate optimal immunity to secondary N. brasiliensis infection. This was related to B cell IL-13 expression, not IL-4 expression. B cell IL-4Rα mediated protection was associated with increased B cell and CD4 T cell IL-13 production. MHCII dependent B cell priming of T cells associated with this effect. Our data also demonstrated a rapid poly-functional antigen processing associated with B cell Myd88 expression.
B cell responses to N. brasiliensis have been suggested to be largely redundant [20]. Both our current study and that of Liu et al (18) demonstrate an absence of B cells per se does not alter host ability to control N. brasiliensis infection. However, we now show that a molecular change in B cell function, such as cell specific disruption of IL-4Rα expression on B cells, significantly impairs host ability to resolve N. brasiliensis infection. These findings also demonstrate important differences between B cell dependent immunity to N. brasiliensis and H. polygyrus. B cell Be2 immunity to H. polygyrus is dependent on B cell IL-4 production, B cell IL-4Rα expression and antigen presentation. As with H. polygyrus, N. brasiliensis re-infection is also dependent on B cell IL-4Rα expression and antigen presentation, however, B cell IL-13 production appears to play a functional role and not IL-4.
The protective B cell response we demonstrate may be independent of antibody and instead mediated through a B effector response. Such responses are particularly important in controlling CD4+ T cell driven immunity [34] via direct B and T cell interactions [35] as well as B cell cytokine production [36]. These B effector responses have an equivalent diversity in immune polarisation as T cells; producing Be1 (TH1) [35], Be2 (TH2) [36] and Breg (Treg) [37], [38] effector B cells respectively. Functionally Be1 cells contribute significantly to immunity to bacterial infections, such as Salmonella [39], [40], [41]. In helminth infections Be2 cells have been demonstrated to be important for immunity to H. polygyrus [21], although humoral contributions also play a significant role [19], [20]. Evidence of Bregs induced by both Heligmosomoides polygyus and Schistosoma mansoni infection have elegantly shown helminth elicited B cell control of allergy [38], [42].
In this study we show that B cells develop a rapid and potent protective response against N. brasiliensis infection. This rapid protection precludes BCR-dependent clonal expansion following antigen exposure. Instead, it appears B cells are capable of responding to antigen via less stringent mechanisms than the BCR, such as direct peptide loading and Toll like receptors [29], [30]. Antigen binding by TLRs is established as an important regulator of B cell function [32]. TLR-dependent B cell responses can increase BCR-dependent antigen presentation [31], B cell cytokine production [39], [40] and play pivotal roles in B cell ability to interact with T cells [33]. These TLR mediated responses to antigen by B cells can be rapid and may not require clonal expansion of B cells [27].
In addition to antigen presentation we also demonstrate IL-4Rα-dependent increases in IL-13 production by endogenous B cells to be associated with control of secondary infection. This along with B cell-dependent induction of IL-13 production by endogenous CD4+ T cell and B cells would provide an important source of IL-13 to activate potential effector cell populations, including epithelial [13], [43], smooth muscle [12], [44] and innate immune cells [24], [45], [46].
In summary this study demonstrates IL-4Rα-responsive B cells playing an important role resolving secondary N. brasiliensis infection. We suggest the protective role played by B cells develops from antigen encounter with TLR driving an increase in CD86 and MHCII dependent interactions with CD4 T cells. This drives increased IL-13 production by CD4 T cells and B cells, facilitating host launching of protective mechanisms against N. brasiliensis infection.
Animals used
In this study the following BALB/c background mice were used: BALB/c, BALB/b, IL-4Rα−/− [described as Il4ratm1Fbb/Il4ratm1Fbb], IL-13−/−, IL-4−/−and μMT. BALB/c background B cell specific IL-4Rα deficient MB1CreIL-4Rα−/lox [described as Il4ratm1Fbb/Il4ratm2FbbTg (Cd79atm1(cre)Reth)] were generated as previously described [26]. MHCII−/−, MyD88−/− and C57BL/6 mice were on C57BL/6 genetic background. Mice were bred and housed in specific pathogen–free conditions at the University of Cape Town, South Africa, and used in accordance with University Ethical Committee guidelines. All experimental mice were sex matched and used between 6–12 weeks of age with appropriate littermate controls of the same generation.
Ethics statement
All studies were carried out under protocol 008/019 approved by the University of Cape Town Faculty of Health Sciences Animal Ethics Committee in accordance national guidelines laid down by the South African Board of Standards.
N. brasiliensis infection
Mice were initially inoculated subcutaneously with 500 N. brasiliensis L3 larvae. At day 7 post infection worms were cleared by treatment with 10 mg/ml Ivermectin in drinking water for 7 days. Mice were then shelved for 21 days prior to a secondary subcutaneous infection with 500 N. brasiliensis L3 larvae. Mice were killed at day 5 post-secondary infection by CO2 inhalation. Adult worm burdens were determined as previously described [12]. Briefly, intestines were removed from infected mice and the lumen exposed by dissection. Intestines were then incubated at 37°C for 4 h in 0.65% NaCl. Intestinal tissue was then removed and the worms in the remaining saline solution counted.
Tissue samples were fixed in a neutral buffered formalin solution. Following embedding in paraffin, samples were cut into 5 µm sections. Sections were stained with periodic acid-Schiff reagent (PAS) in order to visualise goblet cell hyperplasia [15]. The Histological Mucus Index (HMI) was used to quantify PAS positive airway epithelial cells. Sections photographed at 100× were overlaid with a standard grid. The number of grid units containing PAS positive epithelial goblet cells were divided by all units containing epithelial cells to establish the HMI.
Determination of antibody titres
Parasite specific serum antibody levels from infected animals were determined as previously described [15]. Briefly, flat-bottom 96-well plates were coated overnight with 10 µg/ml of N. brasiliensis antigen. The plates were then washed and incubated in PBS containing 2% milk powder v/v for 1 h at 37°C. Following this, the plates were washed, samples loaded and incubated overnight at 4°C. Appropriate biotinylated secondary antibodies were then added following further washing and incubated overnight at 4°C. The plates were then washed, and antibody titres were determined using streptavidin-coupled horseradish peroxidase. The plates were developed with the TMB microwell peroxidase substrate system, and the reaction was stopped with 1 M H3PO4. The absorbance at 450 nm was determined with a Versamax microplate spectrophotometer (Molecular Devices, Germany).
FACS analysis
The expression of surface receptors involved in B and T cell interactions were measured on mediastinal lymph node cells. Essentially, CD40-PE (clone 3/23), CD28-PE (clone 37.51) and TCRβ-biotin (clone H57-597) antibodies were used to detect receptors on CD4+ (clone GK1.5) T cells, while CD40L-PE (clone MR1), CD86-PE (clone GL1) and MHCII-bio (clone M5/114) were used to detect receptors on CD19+ (clone 1D3) B cells in IL-4Rα−/lox and mb1CreIL-4Rα−/lox mice. Biotin-labeled antibodies were detected by streptavidin-APC, anti-FcR (clone 2.4G2) was used to block non-specific binding of immunoglobulins to the FcγII/III receptors and dead cells were excluded from analysis by 7-AAD staining (Sigma). Antibodies were from BD Pharmingen (San Diego, CA). Cells were acquired using a FACSCalibur (Beckton-Dickinson, Ferndale, South Africa) and data were analysed with Flowjo software (Treestar).
Intracellular cytokine staining was performed on mediastinal lymph node cells re-suspended in complete media (IMDM (GIBCO/Invitrogen; Carlsbad, CA), 10% FCS, P/S) at 2.5×107/ml and stimulated with 10 µg/ml of N. brasiliensis antigen and GolgiStop (as per manufacturer's protocol; BD Pharmingen) at 37°C for 4 hours. After re-stimulation, cells were surface stained for CD3 (clone 500A2), CD4 (clone GK1.5) and B220 (clone RA3-6B2), then fixed and permeabilized with Cytofix/Cytoperm Plus (as per manufacturer's instructions; BD Pharmingen). Intracellular staining was performed by staining cells with IL-13-PE (ebio 13a) or appropriately labelled isotype control (eBioscience) [47].
Sorting of B cells for adoptive transfer
Single cell suspensions from spleen were surface labelled with CD19 and B220 antibodies described above, re-suspended at 1×107 cells/ml in media and sorted with a BD FACSARIA cell sorter (Supplementary Figure S3A). The purity of the isolated population was confirmed by flow cytometry, and samples showing <95% positive cells were discarded (Supplementary Figure S3B). Then the isolated B cells from naïve or infected mice were adoptively transferred to naive mice. The cells were re-suspended at 2.5×106 cells/ml in media. Each mouse received 0.5×106 B cells injected intravenously into the tail vein 24 h prior to infection with N. brasiliensis.
In vitro B cell antigen pulsing
Naïve B cells were isolated from a single splenocyte suspension by FACSARIA as described above. Purity was confirmed by flow cytometry, samples showing <95% B220 positive cells were discarded (Supplementary Figure S3). Cells were incubated (pulsed) with 10 µg/ml N. brasiliensis antigen, ovalbumin, LPS or soluble Leishmania antigen for 16 h at 37°C. Cells were then washed 3× in media by centrifugation and then re-suspended in media at 2.5×107/ml. 0.5×106 cells were then transferred intravenously into naïve mice 24 h prior to infection with N. brasiliensis [27], [28].
Generating N. brasiliensis somatic antigen
L3 larvae were washed from filter paper into H2O/50 µg/ml Penicillin+Streptomycin and allowed to stand for an hour during which the larvae settle to the bottom of the container, after which the larvae washed once more in H2O/Pen./Strep and once in H2O. Then the larvae were concentrated into 2 ml of distilled H2O and snap frozen in liquid nitrogen. Following this the preparation was homogenized for 5 to 10 minutes before the whole solution is centrifuged at 10 000 rpm for 10 minutes. The supernatant contains the soluble fraction of the L3 larvae proteins which is measured and standardised using a BCA protein assay (Pierce; Chicago, IL). Antigen was added to the cells in solution at 10 µg/ml.
Values are expressed below as means ± standard deviations and significant differences were determined using either Mann-Whitney U test or ANOVA (GraphPad Prism4).
Supporting Information
Figure S1.
Lung epithelial mucous production is reduced in IL-4Rα−/− and IL-13−/− mice but not in IL-4−/− or MB1creIL-4Rα−/lox mice. IL-4Rα−/−, IL-4−/−, IL-13−/−, MB1creIL-4Rα−/lox and IL-4Rα−/lox mice were infected for 5 days post-secondary N. brasiliensis infection. Pulmonary mucus production was established by PAS staining (Figure 1 and 2). The Histological Mucus Index (HMI) [15] was used to quantify the numbers of PAS positive epithelial cells.
Figure S2.
Spleen B cell IL-13 production is reduced in MB1creIL-4Rα−/lox mice. Naïve MB1CreIL-4Rα−/lox and IL-4Rα−/lox mice were infected with N. brasiliensis. Splenic B cell IL-13 responses at day 10 post infection were established by intracellular FACS staining in B220+CD19+ populations.
Figure S3.
Gating strategy for isolation and establishing purity of B cells. The purity of B cells was established by flow cytometry using the gating strategy shown (A). Purity was over 95% in all cases (B). Briefly, lymphocytes were identified according to forward scatter vs. side scatter profile. CD11cneg, CD3neg, CD4neg and GR-1neg and B220+CD19+ cells were then isolated and used for further analysis..
Figure S4.
Transfer of naïve B cells does not confer protection against N. brasiliensis infection. B cells isolated from naïve BALB/c mice were pulsed with antigen and transferred into naïve BALB/c mice. Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were then established at day 5PI.
Figure S5.
B cell IL-13 cytokine responses at 1 day post N. brasiliensis infection. Mice were infected with 500xL3 N. brasiliensis larvae and killed 1 day post infection. Splenic B cell IL-13 production was established by flow cytometry.
Figure S6.
B cell mediated immunity to N. brasiliensis is antigen specific. B cells isolated from naïve BALB/c mice were pulsed with antigen and transferred into naïve BALB/c mice. Mice were then infected with 500xL3 N. brasiliensis larvae and worm burdens were then established at day 5PI. Antigen specific protection by B cells was established by pulsing B cells with N. brasiliensis, L. major or Ova antigens or LPS then adoptively transferring into naive BALB/c mice.
The authors would like to thank the animal facility staff, the genotyping staff, Lizette Fick and Ronnie Dreyer for their excellent technical assistance.
Author Contributions
Conceived and designed the experiments: WGCH MGD FB AJC. Performed the experiments: WGCH MGD JCH NN HN SB MK FK BD AFC HJM. Analyzed the data: WGCH MGD JCH NN. Contributed reagents/materials/analysis tools: WGCH FB AJC. Wrote the paper: WGCH MGD FB AJC.
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10 reviews | 2.8 out of 5
I bought this bike only a week ago for its price. I wanted a mountain bike that I didn't have to pay $150 for it. This was my choice. I have ridden it a couple of times, but nothing big. Just a visit to the store 1 mile away. The seat was a bit hard and uncomfortable, so I bought a gel seat. The back brake touched the handlebar when I braked, but that was taken care of. Overall, its not a really bad bike if you just want to use it for rides not so far away. I still haven't tried riding for a couple hours or on mountain trials.
p.s. my bike is a different color but the same model and the gel seat is a bell gel fusion seat.
The bike is a nice size and very pretty, however, the seat is HORRENDOUS!! I had to go the bike shop and buy a new seat that cost $30.00. Now that I am ready to ride the bike the chain is loose. It seems that I am spending more time and money getting it ready to ride than riding it. I am not very happy with this product.
I've only owned for 2 weeks and have gotten to ride once..Weather hasn't been great for riding a bike..lol I would highly recommend this bike.I suffered a heart attack over 6 months ago and I needed an affordable bike and a mountain bike where I live the only place to ride is on a gravel road.I've rode and I have only one down fall with the bike the seat will be painful if rode very far.I will be replacing the seat.Thanks for a reasonable and afforable bike!
Hard to ride. Brakes broke the SECOND DAY!
A fun ride !!!
Although the bike I have is a different color, this is the same one. I have had my bike for only a month and have rode it 4 times. You have to pull the brakes all the way to the handle to get them to catch (definately not good on a hill). Had NO speed whatsoever.
When I went to get on it tonight to ride, the left peddle fell off & we couldn't get it put back in because the bolt was stripped and whoever put it together...didn't completely screw in all the screws. They were all halfway sticking out & the saleslady said that's how they were suppose to be!
We are returning the bike to Super WalMart this week!!!!
We just bought this bike today and had a test run and it does NOT work with a child seat on the back. There was not enough room and my son's feet kept getting in the way of my peddling.
I bought this bike for my daughter (age 12 and 5'3" tall). She loves it and rides it everyday! We put a gel seat cover on it to make it a little more comfortable.

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Category: pottery
Back to our roots
By Valerie DeLaCruz on August 19, 2013 at 9:00 AM
There's a new design trend embracing a more primitive style, celebrating hand-carving and rich warm colors of the savannah. At Homegoods, I found these beautiful woven plates that could double as …
Hippity Hoppity Pottery Barn
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Looking to get in the mood for Easter and Spring? Pottery Barn is featuring a wonderful line of of bunny rabbit-themed ceramic and serving pieces. I love this jaunty fellow, kickin' back and …
Country accents and folk art
By Valerie DeLaCruz on February 1, 2013 at 9:08 AM
It's so cold here in the winter; it makes me want to curl up by a cozy fire. New England and colonial style calls to mind a hearth and warmth. And that 's what you can add when you're selecting some …
Hand-thrown Hilborn Pottery
By Valerie DeLaCruz on January 2, 2013 at 9:10 AM
Pearl Grant Richmans features a beautiful collection of Hilborn Pottery Design. Owned and operated by Rick and Nancy Hilborn, the pottery shop has been producing Canadian hand-made pottery since …
October decorating
By Valerie DeLaCruz on October 10, 2012 at 9:10 AM
Looking for some items to freshen up your decor with a fall theme? Try Pottery Barn for some clever and chic pumpkin and gourd inspired accents: With just the addition of some new napkins and charger …
On the trail to Adirondack Trails
By Valerie DeLaCruz on October 3, 2012 at 9:14 AM
In the quaint town of Bolton Landing, there are several boutiques, including one of my favorites, the Adirondack Trails Store. Walk up the welcoming porch and into a store that has home decor items …
Find your home's essence at Homessence
By Valerie DeLaCruz on August 15, 2012 at 9:10 AM
It's not often we get a new home decor store, and even more rare to get one with such a great mix of quality, unusual items. I discovered one such gem right on Broadway in Saratoga during a recent …
Strolling Village Artisans
By Valerie DeLaCruz on June 13, 2012 at 9:15 AM
Up on Art Alley in Ballston Spa, there's a great cooperative gallery called Strolling Village Artisans. A combination shop and gallery, each room features a different craftsperson or artist's work. …
Mix it up, be eccentric
By Valerie DeLaCruz on March 30, 2012 at 9:34 AM
…that's the theme of the Artcentric Gallery at 266 River Street in Troy. A combination of affordable art, local craftsmen, fair trade items and vintage furnishings, there was something interesting …
Throw a pot, break the mold
By Valerie DeLaCruz on March 26, 2012 at 9:30 AM
At the Market Street shops on River Street in Troy, there is a wonderful pottery place, The Broken Mold Studio. Here you can find original hand made ceramic pieces by local artisans. Lovely platters, …


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Vendégek száma4
Hálószobák száma1
Méret75 m²
Floor No.(with elevator)
- Családi / Gyerek barát
- Internet/WiFi
- Konyha
- Légkondicionálás
- Mosógép / szárítógép
- Alkalmas rendezvényekre
- Pezsgőfürdő
|STRATIGOU MAKRYGIANNI, 11742 ATHENS, Görögország
|Ingatlan típusa
|Ingatlan azonosító
|Acceptance rate
|Response time
|A few hours
|Calendar updated
The Space
Apartment is accessed by elevator
A luxurious apartment with
1 veranda with unique view of Acropolis
1 bedroom with double real bed and view
1 living room with double quality sofa bed
1 fully equipped kitchen
1 bathroom with washing machine
Bed linen and towels are provided
Free Wi-fi, Air condition, TV, hair dryer, etc
We can provide baby cot if you want to travel with kids.
-- Apartment with unique view of the Acropolis
-- Sleeping,relaxing,dining under the shadow of Parthenon
---All rooms are Air Conditioned
-- Apartment is Opposite Museum , metro and Acropolis
-- No Hotel can offer such view and proximity at this price .
-- Babies under 1 year old free of charge
- Airport /port transfer is available upon request.
- We offer basic ingredients to prepare by yourself your breakfast (coffee, tea, sugar, cereals,)
- We are the first, awarded by AIRBNB, Super-host in Athens.
- No extra cost, and no need for safety deposit
A 65 sq m. luxurious, comfortable,clean, apartment with a superb 20 sq m. veranda, with fantastic view over timeless Parthenon temple. A sense of luxury in the most historic place of all times, on the famous pedestrian-street, just opposite the ancient rock of Acropolis.
PLEASE NOTE, as the apartment is situated on the pedestrian street ,parking is not allowed in this area. There are private parking stations in 100 meters.Streets are full of tourists all year long. No recycling in this area .
We try to be environmentally friendly , so bed sheets/towels are washed with ORGANIC detergent .
Guest Access
The entire apartment and its amenities are at your exclusive use.
There are 2 options to reach the apartment.
1) Private transfer (extra cost). Arriving or leaving after midnight? No problem!
2) Arriving by your self and meet you at the apartment. This option is available for arrivals between 11 in the morning till 8 in the night.
Interaction with Guests
By making a reservation with us, you are getting access to many suggestions for tours or accommodations, in many islands and places of interest all over Greece.
The Neighborhood
Check the photos to see the superb view we are offering. You are in the heart of historical Athens! You are indeed opposite the Acropolis hill. The area consists the ideal solution to those who want to explore on feet all surrounding places of historic interest such as; Plaka, Monastiraki, Thissio, the ancient market of Athens, Herodion theatre, as well as the museum of Acropolis which is only 80 meters away. Furthermore, within 2 minutes walk from the house you can choose from a vast variety of restaurants , banks, bakery, pharmacy e.t.c.
Getting Around
BY CAR - 37' minutes. If you like we can arrange pick up service at a friendly price
BY METRO - Minimum 2 hours (including waiting 30 minutes for the metro and walking). Athens centre IS QUITE FAR from the airport. ACROPOLIS metro stop is opposite the apartment
BY CAR - 13' minutes. If you like we can arrange pick up service at a friendly price
BY BUS & METRO - Minimum 45' min (depending on the island, walking to the ferry can add up to 30 minutes extra time).
You are opposite Acropolis and its museum!
You are in the heart of historical Athens. All major sites are within 5 minutes walking distance
Other things to note:
Families friendly, gay friendly, lesbian friendly, single women travel friendly.
After your booking, we are sending to your email, a document with all the different options of reaching the apartment and a map of the area.
Be prepared to experience Athens the authentic way...the way we do!
1) Trash
Guests are requested to deposit their trash regularly (and certainly prior to their departure) in the grey colored trash collection containers in the street. Please recycle. The blue containers in the street are for recycling materials.
2) Kitchen
We expect from our guests, prior to their departure to clean their dishes and leave the kitchen as clean as they found it. In case that you do not wish to clean your dishes or you do not have time to do it, you can leave 14 euros in cash in the kitchen for the cleaning lady.
3) Toilet.
Thank you for helping us save the environment and the Greek seas. No paper towels, kleenex or feminine products to be flushed in toilet.
With every stay regardless the length of stay, we offer 2 rolls of toilet paper
4) Respect the neighbors. Please restrain from making loud noises between 02.30 pm-5.30 pm and 11.00 pm -08.00 am.
5) This is a NON smoking apartment. Smoking is allowed only in the Veranda.
6) Towels are provided but they are not to be used as a bath mat , or to remove make up.
7) Please lock with the key the entrance of the building after 10 in the night.
By making this reservation, you accept that this not a hotel. This is a house and you are a guest. The host cannot be held responsible for any accidents or injuries that occur to guests on the premises.
Guests are held responsible for their own actions if they break the law. Use of drugs inside the apartment is prohibited. The Greek law covers this agreement and any civil difference should be held in Athenian courts.
|Weekly rate for up to 2 guests
|895 €
|Monthly rate for up to 2 guests
|3084 €
|Ár >2 vendégre
|22 € /fő/éj extra
|Maximális tartózkodás
|3 hónap
|Érkezés legkorábbi időpontja
|Check-in before
|Távozás legkésőbbi időpontja
|Lemondási feltétel
Very nice house for a group of friends or a big family with many conveniences such as many bathrooms, rooftop, terrace. The kitchen is well equipped in general, but a few utensils are lacking. The house is in perfect condition and well built for keeping it cool on hot days. The location is very good, near beaches and Naousa town where bars and restaurants can be found. We rented cars and there is sufficient parking space in the driveway for 2-3 cars and motorbikes. The island is small so getting to places is quite quick no matter where you are.
Splendid location for visiting Athens and afterwards cooling down at the beach. Service and response of Helena (and her team) was always accurate and meeting expectations. Day tour to Peleponnesos was great. Fam in t Veld
Our stay at this Appartement was great! We had some Problems with the fridge and the a/c in the beginning but after just one call Helena and Vasilis did all the could and so we got a new Fridge and a "new" a/c within only a few days!
The Appartement is great, of course not the most modern but the view and the Balcony makes you forget what's inside, and so most of the time we spent in the Appartement we were on the balcony enjoying the view to the sea.
The Bolivar Beach and Bar is amazing also and ist just across the street from the Appartement. We also found some very nice small Restaurants more in the "middle" of the town, where the staff was very friendly.
We would really recommend that Appartement, cause it has got all you Need for a calm and Relaxing Holiday.
We enjoyed a lot our stay. Chryssa was so kind with us when we arrived. The house was nice and near to the city centre.
Everything was perfect!!!! The apartmet really confortable, and near to the tram which is directly connected with Santagma Square and the metro. We really appreciated our vacation in Athens and surely we will come. Thanks to Helena and a big hug to Cryssa, beautiful person, always available and really kind. We could't choose better than this. Thanks
We stayed at this lovely appartment one night with my boyfriend, my brother, his wife and their 11 months old baby girl. It was perfectly located for a brief visit of Athens! Very cute and well maintained appartment in a quiet residential area 10-15 minutes to the Acropolis!
We will be back;)
Wow just what we needed for a 6 day stay in Athens, 2 bedrooms with good beds, small kitchen with everything you need, bathroom adequate, lounge area large with huge verandah. Restaurants and supermarkets at your door step. The base of the Acropolise only a 5 minute walk. Greeting and key hand over completed with ease. A great place to stay.
Our family of 5 very much enjoyed the apartment, overall. We were met promptly by a kind and enthusiastic associate who got us settled in. The apartment was close to all the places we wished to visit (by foot)! The space was roomy, clean , and quiet. The kitchen was well stocked with dishes. The terrace was very large, but we wished that there was an awning, as you could not be out on it in the direct sunshine. Also, all 4 deck chairs were broken (seats ripped and could not bear weight). There were travel books available for our use, and were appreciated. Would recommend the apartment!
The area of Helena's apartment is quiet and safe.
The location is perfect - straight connection (several tram station) with the center of the city and at the same time with the beaches at the seaside.
There is a park at the same street and large shopping area in the distance of one tram station.
The apartment is very clean and comfortable.
Full information is clearly given for the guests - concerning all the things a tourist is looking in the city.
The host is very friendly, open and offers her sincere help. In fact you fill like under your mother's care.
When visiting Athens again I will try to come back to Helen'a apartment.
We were very warmly welcomed at the airport and everything was fine during our stay. The apartment is quite spacious and can easily accommodate 3 couples. The sights were easily accessible on foot. Also shopping were very close. The balcony was a bit narrow but still offered seating for 5 people. The terrace didn't use, The TV had only 3 programms but because the most of the time we were in the city and on the beach, this did not play a role. All in all, we can recommend the apartment very much.
- Minőségbiztosított szállás
- Minden házigazda hitelesítésre került telefonon keresztül
- Biztonságos fizetés SSL titkosításon keresztül


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What you get
- Entry for One, $10 to Spend on Merchandise, Medal, Beer & More
- Obstacle- and Zombie-Filled 5K or 15K Course
- Stick Around for the Post-Race Survival Party
- Following Purchase, Register Voucher & Select Wave Time Online by July 23
You may have heard of dead men walking, but with this offer, they'll be chasing. Get ready to flee from the zombie horde when you sign up for the Zombie Race -- either 5K or 15K -- in Riverhead on Saturday, September 27.
Walk, run, and crawl for your life through adrenaline-inducing obstacles like a giant mud pit, a cargo net, and more as the blood-thirsty zombies trail you. If you make it to the finish line with at least one of your three health flags, you can proudly call yourself a Zombie Race Survivalist. Every entrant will receive a sack pack, $10 merchandise credit, one beer (for those 21+), three health flags, racing bib, finisher's medal, and entry to the post-race party with food, and live entertainment.
Choose from the following offers:
• $42 ($85 value) for one entry in the 5K
• $52 ($105 value) for one entry in the 15K
what to bring
• Photo ID
• Cash for food, beer, gear, and post-race activities
• A towel and a change of clothes
• Zombie costume
• Zombie makeup
Fine printVOUCHER IS NOT YOUR TICKET; ONLINE EVENT REGISTRATION REQUIRED BY September 26, 2014 • REFUND POLICY: This event is nonrefundable. No full or partial refunds are given to customers who miss an event. In the event of a cancellation, your voucher will be fully refunded • Each voucher is good for 1 person • Excludes $10 insurance fee • Voucher value reflects pricing without any early registration discounts • Must be 21+ with valid ID to consume alcohol • Alcohol not discounted more than 50% • The deal is valid only for tickets on the date[s] of the event as listed and may not be exchanged or redeemed for any other dates • Entire value must be used in a single visit
Other conditions apply
Except where noted in the fine print:
• Cannot be combined with any other offer or promotion
• Tax and gratuity are not included
• Merchant is responsible for the quality of the products or services provided to you at redemption
• LivingSocial Terms and Conditions - https://m.livingsocial.com/terms
Entry Package to Zombie Race
times for
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Pick-Up Locations: West 45th and 11th Avenue | 85 Delancey Street (Between Ludlow & Orchard)
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VIP Ticket to Señor Frogs Drag Brunch with Unlimited Cocktails
Señor Frogs - NYC
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Spa Castle - New York
All-Day General Spa Admission + 4-Season Grand Spa Pools
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Prix-Fixe Dinner for 2 with Bottle of Wine
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Admission or Membership to Children's Museum
Northstar 2 Fishing
3-Hour Fluke/Flounder Fishing Trip
Go City Card
Up to 45% off Top New York City Attractions
Nino's Restaurant
3-Course Surf and Turf Dinner for 2
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Reality Check
Installed between hose and sprinkler, the WaterWatch reveals how many gallons of water you've consumed in an irrigation session. Like a bathroom scale or bank statement that tells no lies, this simple device might inspire you to cut back.
Approximately $6; EFI
Ask TOH users about Yard and Garden Tools


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Reload the page and try again.
The university grants free transportation around campus and around half the city.
very slow but I guess its due to the number of stops made during each route.
transportation is convience for getting to class or around campus
Can not really rate this because we do not have it.
Ok because, the bus is free for student just show I.D., however, the wait is long just know when the bus comes, because if you miss it may be another 30-45 minutes if not longer. Freshmen, aren't allowed a vehicle their first year but after that its okay. the bus line runs throughout the city and to some places on the outskirts too.
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updated 08/25/2015 AT 8:16 PM ET
•originally published 02/20/2014 AT 12:10 PM ET
Matthew McConaughey has called his Dallas Buyers Club transformation a physical and spiritual journey. But there was a single moment while playing AIDS activist Ron Woodroof that he says felt like "a soul punch."
Appearing on a new episode of Inside the Actors Studio that airs Thursday night (8 p.m. ET) on Bravo, the Oscar nominee discusses a line that will stay with him forever.
"There's a scene in this film where Ron goes, 'I'm dying.' Those two words, 'I'm d-y-i-n-g,' I didn't expect it but it just … hit me and it floored me," McConaughey tells host James Lipton.
Up for Best Picture at the 2014 Academy Awards on March 2, Dallas Buyers Club follows HIV-positive outlaw Woodroof as he smuggles medicine into Texas and fights pharmaceutical companies peddling a dangerous AIDS treatment.
McConaughey famously shed almost 50 lbs. for the role. Though he has since regained his muscular figure, the actor says he will always empathize with Woodroof's realization that he was approaching death.
"Saying those worlds floored me in a way that I think definitely grounded me since then in a different way than I was before," he says. "Maybe it's my own mortality, but it was a moment personally for me, that I found through Woodroof, that's never gonna leave me."
Watch a clip below, and catch the full interview Thursday on Bravo's Inside the Actors Studio.


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Container Service
Green Earth Metal Recycling provides on-site container service for industrial accounts, aluminum can collection centers and others.
We Purchase Ferrous And Non-Ferrous Metals
We buy:
Ferrous (Steel Scrap)
Metal Price Quotes Over The Telephone & Online
Please call us for a quote on all your scrap ferrous and non-ferrous metals, obsolete computer hardware and peripherals.
214.428.3702; firstname.lastname@example.com
We Do NOT Purchase Environmentally Harmful Products
Products we do not buy:
Exceptional Customer Service
Come and experience our exceptional customer service. Green Earth Metal Recycling's first priority is to provide you with better than expected service. We customize service solutions for your recycling needs. We are open 7 days a week:
Monday-Saturday 7:00AM - 7:00PM
Sunday 7:00AM - 4:00PM
Computer Hardware And Peripherals
Green Earth Metal Recycling purchases recyclable computer hardware and peripherals.
A certificate of scrap metal destruction will be provided upon request, which states that all data on storage devices has been electronically wiped clean or physically destroyed.


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The Blog
The Joe Rogan Experience #804
Wherein Sam and Joe talk for 4.5 hours…
The Multiverse & You (& You & You & You…) Podcast Transcript
Known as "Mad Max" for his unorthodox ideas and passion for adventure, Max Tegmark's scientific interests range from precision cosmology to the ultimate nature of reality, all explored in his new popular book Our Mathematical Universe. Tegmark is a professor of physics who has published more than two hundred technical papers and been featured in dozens of science documentaries. His work with the Sloan Digital Sky Survey on galaxy clustering shared the first prize in Science magazine's "Breakthrough of the Year: 2003." For more information about his work, please visit his MIT website and the Future of Life Institute.
The Limits of Discourse As Demonstrated by Sam Harris and Noam Chomsky
For decades, Noam Chomsky has been one of the most prominent critics of U.S. foreign policy, and the further left one travels along the political spectrum, the more one feels his influence. Although I agree with much of what Chomsky has said about the misuses of state power, I have long maintained that his political views, where the threat of global jihadism is concerned, produce dangerous delusions. In response, I have been much criticized by those who believe that I haven't given the great man his due.
Last week, I did my best to engineer a public conversation with Chomsky about the ethics of war, terrorism, state surveillance, and related topics. As readers of the following email exchange will discover, I failed. I've decided to publish this private correspondence, with Chomsky's permission, as a cautionary tale. Clearly, he and I have drawn different lessons from what was, unfortunately, an unpleasant and fruitless encounter. I will let readers draw lessons of their own.
Very Bad Wizards Interview #2 Sam Harris, David Pizarro, and Tamler Sommers keep talking
Episode Info
Sam Harris gets back in the VBW ring for another round on moral responsibility, ethical theories, and the grounds for our obligations to other people. Are we at a genuine stalemate when it comes to blame and desert? Is Tamler a closet consequentialist? Is Sam a closet pluralist? Why is Dave such a big Wagner fan? Plus, Twitter shaming: what is it good for? Settle in, get comfortable, pour yourself a drink, you're in for the long haul on this one.
On Being Right about Right and Wrong An Interview with Michael Shermer
Michael Shermer is the Publisher of Skeptic magazine, a monthly columnist for Scientific American, the host of the Skeptics Distinguished Science Lecture Series at Caltech, and a Presidential Fellow at Chapman University. His latest book is The Moral Arc: How Science and Reason Lead Humanity Toward Truth, Justice, and Freedom.
Michael was kind enough to answer a few questions by email:
Can We Avoid a Digital Apocalypse? A Response to the 2015 Edge Question
It seems increasingly likely that we will one day build machines that possess superhuman intelligence. We need only continue to produce better computers—which we will, unless we destroy ourselves or meet our end some other way. We already know that it is possible for mere matter to acquire "general intelligence"—the ability to learn new concepts and employ them in unfamiliar contexts—because the 1,200 cc of salty porridge inside our heads has managed it. There is no reason to believe that a suitably advanced digital computer couldn't do the same.
It is often said that the near-term goal is to build a machine that possesses "human level" intelligence. But unless we specifically emulate a human brain—with all its limitations—this is a false goal. The computer on which I am writing these words already possesses superhuman powers of memory and calculation. It also has potential access to most of the world's information. Unless we take extraordinary steps to hobble it, any future artificial general intelligence (AGI) will exceed human performance on every task for which it is considered a source of "intelligence" in the first place. Whether such a machine would necessarily be conscious is an open question. But conscious or not, an AGI might very well develop goals incompatible with our own. Just how sudden and lethal this parting of the ways might be is now the subject of much colorful speculation.
The Very Bad Wizards Interview #1 Sam Harris, David Pizarro, and Tamler Sommers talk (and then talk some more)
0:00-47:00—Intro and costs and benefits of religion
47:00-1:17:00—Drugs, the self, free will
1:17:30-end—Blame, guilt, vengeance, moral responsibility
David Pizarro is an associate professor in the Department of Psychology at Cornell University in Ithaca, NY. His primary research interest is in how and why humans make moral judgments, such as what makes us think certain actions are wrong, and that some people deserve blame. In addition, he studies how emotions influence a wide variety of judgments. These two areas of interest come together in the topic of much of his recent work, which has focused on the emotion of disgust and the role it plays in shaping moral, social, and political judgments.
Tamler Sommers is an associate professor in the Philosophy Department at the University of Houston with a joint appointment in the Honors College. He is director of the Honors minor Phronesis: A Program in Politics and Ethics. His research focuses on issues relating to moral responsibility, criminal justice, honor, and revenge. Sommers is the author of two books: Relative Justice: Cultural Diversity, Free Will, and Moral Responsibility (Princeton, 2012) and A Very Bad Wizard: Morality Behind the Curtain (McSweeney's, 2009). He received his PhD in Philosophy from Duke University in 2005.
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Efficient Wireless Multicast Retransmission Techniques Based on Multiple Coded Packets
Through wireless multicast transmission various users can receive the same information which is transmitted at the same radio resource. A resource can be understood in various ways such as a time-slot, a particular frequency or a spatial location. This form of transmission is very useful, for example, in the digital TV application, where several users of a particular cell generate a demand for data on the same channel. In the case of UTRAN and its evolution, the multicast services are specified by the MBMS standard, which defines resource allocation and transmission procedures specific for multicasting. One particular bottleneck of wireless multicast services is the retransmission of erroneously received packets.


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Type: Posts; User: Suhas Dhoke
Hello Rashmi
$address = 'email@example.com';
$txt = preg_replace('/\.[^@]*$/', '', $address);
echo $txt
Try with giving the absolute path of JS files, instead of relative.
For Auto-Increment, the field should be the Primary-Key. You can use it through sql statement or by selecting the "auto_increment" from the "EXTRA" drop-down.
You can also set the starting number...
$term = mysql_real_escape_string($_GET['search_term']);
mysql_query("SELECT * FROM table WHERE field LIKE '%{$term}&'");
// ....
If you want to search after from first three letters...
You did a very silly mistake.
Actually, you wrote the query, but you didn't execute it.
In line nos: 15 and 81, use the function mysql_query() to execute the query.
And this will...
Means, do you want to scrolling the text from beginning after reaching the end point ?
Try after commenting the line:
if(pos < 0 - obj.offsetHeight+130) return;
GET and POST are the methods of form.
If you selected the form method as POST then the you can collect the form's data in $_POST['form_field_name'].
The $_GET works only when, if you set it as...
I am not getting an error string back, but it won't let me log in. Now all it does is cycle me to the login.php?login_error=1 page.
B'coz, You are checking the ...
Try to use "OR" instead of "AND" from the query.
The error is occurs coz, the query doesn't contain anything..
Why not you try $_POST var, instead of $_REQUEST., where u can get all the checked values at a time...
Otherwise, you can use the...
No, i meant this..
foreach($_REQUEST['current'] as $key=>$value)
else $checkValue="N";
You wrote the query in the foreach() loop, but executing it outside of loop. Hence, the query is fired only once, for the last loop iteration..
Try to execute it in the same loop.
You can also use this..
var string = document.getElementById('txt').value;
chars = string.split(' ');
if (chars.length > 1)
alert('Space(s) found in the string.');
Use the <IMG> tag instead of Anchor Tag (<A>) for displaying the image, instead of a link.
Here is a test script, where the user can enter only numeric value in the text field.
The validation will apply on submitting the form.
function checkValidQty(f)
I have modified your function MM_goToURL(), to pass the grand total value as the query string.
And that value, you will get to the next page.. But be sure, the next page must be the php page (php...
You are trying to get the value of "total" by using the $_GET.
If you are passing the total cost in GET, then there is no need to add an additional '$' sign
against the field name..
try to use...
Here is the sample script, which used to calculate the total number of chars, words and lines in the TextArea field.
<script type="text/javascript">
function counts()
var string =...
Here is the code, which u want..
Using the JS, adding a new row.. by clicking on the Button.
var row_counter = 2;
function add_new_row() {
I had created a script for,
have a look... do u wanna the same ?
var romanList = 'I,II,III,IV,V';
just for the confirmation.. .
Are u passing the correct *server_username* and *server_password* and
there is no need to add single quote ( ' ) around the Table_Name.
Remove it, if it is...
And, one more thing.. Do u passing the valid *username* as argument in checkUsername() function ?
Give me the code, what u write.. .
First of all, the connection was not established, because, you are using the variables
for connecting to the database, but they are not defined.
Therefore it displays the blank page.


text:
"Retail has always been about theater, excitement and fantasy." — Marigay McKee, president of Saks.
"Our e-commerce heart beats as loud as our fashion heart." — Munich's Theresa boutique managing director Jens Riewenherm.
"We hadn't realized how enormous the potential was. The volumes we needed to buy to satisfy customer demand were so much greater." — Ruth Chapman, co-founder of U.K. fashion mini-chain Matches, on their growing e-commerce presence.
"There will only be 48 to 60 of each piece, and I'm going to sign each one," Abboud said. "And then we won't make them again." — Joseph Abboud on introducing a limited-edition collection at Men's Wearhouse this fall.
"Please remember when I come to borrow some dresses, I did you this favor." — Alexa Chung, who hosted "The Future of Fashion" at the Fashion Institute of Technology.
"The challenge you see in traditional brick-and-mortar — and you're starting to see this online as well — is everyone has the same thing." — Fashion consultant Roopal Patel on the growing e-commerce presence of boutique retailers.


text:
I am new to WordPress and learning a beginner in CSS.
I installed the theme Oxygen and I am trying to edit the CSS. However, when i make changes to the CSS through the editor they don't know up. Can't change the color, positioning, size of images...anything.
So far I have:
1.changed the ftp to 774 in order to edit the css.
2.downloaded a css editor plugin which allows me to edit the css bit by bit. These changes show up on my site, however the actual CSS goes away after I save and x out
3.I have tried deleting cache on my wp site and deleting my browsers cache.
4. I then went into my php to see if I could edit the size of images or anything there...and that doesn't show up either.
Nothing seems to be working. Does anyone have advice?