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Published in Oncotarget V8I53, Oct 31, 2017
Immunization with recombinant rabies virus expressing Interleukin-18 exhibits enhanced immunogenicity and protection in mice
Weiwei Gai, Wenwen Zheng, Chong Wang, Gary Wong, Yanyan Song and Xuexing Zheng _
Weiwei Gai1,2,*, Wenwen Zheng1,*, Chong Wang3,*, Gary Wong4, Yanyan Song1 and Xuexing Zheng2
1College of Veterinary Medicine, Jilin University, Changchun, China
2School of Public Health, Shandong University, Jinan, China
3State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
*These authors have contributed equally to this work
Xuexing Zheng, email: [email protected]
Keywords: rabies virus, IL-18, immune responses, vaccine, antibody titer
Received: March 03, 2017 Accepted: July 18, 2017 Published: September 16, 2017
Several studies have shown that interleukin-18 (IL-18) plays an important role in both innate and adaptive immune responses. In this study, we investigated the pathogenicity and immunogenicity of recombinant rabies virus expressing IL-18 (rHEP-IL18). Experimental results showed that Institute of Cancer Research (ICR) mice that received a single intramuscular immunization with rHEP-IL18 elicited the highest titers of serum neutralizing antibodies and the strongest cell-mediated immune responses to prevent the development of rabies disease, compared with immunization with the parent virus HEP-Flury. Mice inoculated with rHEP-IL18 developed significantly higher IFN-γ responses, increased percentages of CD4+ and CD8+ T-lymphocytes compared to HEP-Flury. Flow cytometry results show that rHEP-IL18 recruited more activated T- and B-cells in lymph nodes or peripheral blood, which is beneficial for virus clearance in the early stages of infection. A higher percentage of mice immunized with rHEP-IL18 survived wild-type rabies virus (RABV) challenge, compared to HEP-Flury mice. Our results show that rHEP-IL18 is promising as a novel vaccine for RABV prevention and control.
Rabies is a fatal zoonotic infectious disease caused by rabies virus (RABV) and afflicts nearly all mammalian hosts [1, 2]. The main feature of RABV is neuroinvasiveness, which refers to the ability to invade the central nervous system (CNS) from peripheral sites. Once clinical signs are present in infected humans or animals, rabies is almost 100% fatal [3]. According to the World Health Organization, there are approximately 55,000 human deaths each year worldwide, including 3000 cases in China [4–6]. Dogs and cats are the major reservoir of rabies and most human cases occur by bites from rabid dogs in developing countries in Africa and Asia [7–9]. Vaccination is the most effective way to prevent and control rabies in animals and humans [10–12]. In contrast to other RABV vaccines, most live-attenuated vaccines replicate very quickly and express large amounts of glycoprotein (G), thereby inducing strong adaptive immune responses that result in virus clearance [13]. Live-attenuated RABV vaccines and recombinant live vaccines have already been licensed, such as a recombinant vaccinia virus expressing the RABV G protein (VRG) and a live avirulent RABV, SAG-2, particularly for wild animals [14–17]. These vaccines provide effective immunization with a single dose and has practical, economical, and logistical advantages over conventional multi-dose vaccines with respect to the goal of eradicating rabies worldwide [16, 18–21]. In addition, live-attenuated RABV vaccines can induce immune responses to clear virulent RABV from the CNS and there is the possibility that such vaccines could serve as the foundation for the treatment of early stage human rabies infections [22, 23].
Previous studies show that interlukin (IL)-18 is a pleiotropic cytokine that plays an important role in both innate and adaptive immunity, with an ability to produce high amounts of IFN-γ [24, 25]. Its function is mainly reflected in the enhancement of T cell-mediated immunity [26], but IL-18 is a unique cytokine that can enhance innate immunity and both Th1- and Th2-driven immune responses [27, 28]. IL-18 enhances T cell-activated monocytes to induce IFN-γ production, which is dependent on the activation of intracellular NK-κB and PI3-kinase pathways. But IL-18, without help from IL-12, induces Th2 cells by induction of IL-4 production from naive T cells. Thus, IL-18 has the potential to stimulate both Th1 and Th2 responses [28]. Therefore, IL-18 is an interesting candidate for the modulation of immune responses.
In this study, the gene for IL-18 was cloned into the HEP-Flury cDNA clone. The recombinant RABV, rHEP-IL18, was rescued and used to immunize mice in comparison with HEP-Flury. Our results show that rHEP-IL18 is less pathogenic and more immunogenic compared to HEP-Flury in mice, with recruitment and activation of higher numbers of T and B cells, and provided enhanced protection against challenge with wild-type RABV.
Generation and characterization of recombinant attenuated RABV expressing murine IL-18 (rHEP-IL18) in vitro
The murine IL-18 gene was cloned into the HEP-Flury genome, as shown in Figure 1A. The insertion of the IL-18 gene was confirmed by sequencing the infectious clone and the recombinant virus, designated as rHEP-IL18, was rescued in BSR cells. In order to characterize rHEP-IL18 in vitro, viral growth kinetics were examined in BSR and NA cells. As shown in Figure 1B and 1C, a significant difference in values was not observed between recombinant viruses and the parent virus HEP-Flury, indicating that viral replication was not affected by the insertion of IL-18 gene. Moreover, the expression of IL-18 was detected by Western blotting. As shown in Figure 1D, one target band with molecular weights of 20 kDa corresponding to the positive control was detected, demonstrating that mouse IL-18 were expressed in the rHEP-IL18 infected cells, while no IL-18 was expressed in cells mock-infected or infected with HEP-Flury. In addition, the expression of IL-18 was measured by ELISA. As shown in Figure 1E, murine IL-18 was expressed in a dose-dependent manner in BSR cells infected with rHEP-IL18.
Figure 1: Construction and characterization of recombinant RABV expressing IL-18 in vitro. (A) Schematic diagram for the construction of rHEP-IL18. The HEP-Flury vector was constructed from HEP-Flury strain by adding BsiWI and NheI sites between the G and L genes. Mouse IL-18 genes were cloned between the G and L. (B) Virus growth curves in BSR cells (B) and NA cells (C). Cells were infected with HEP-Flury or rHEP-IL18 at a multiplicity of infection (MOI) of 0.01. Viruses were harvested at 1, 2, 3, 4, 5 and 6 dpi, and viral titers were determined as described in Materials and Methods. All titrations were carried out in quadruplicate. (D) The expression of protein IL-18 was determined by Western blotting analysis. The cells were collected and lysed for Western blotting after BSR cells were infected with rHEP-IL18 or HEP-Flury for 48h. Recombinant pcDNA3.1-IL-18 was used as a positive control. (E) The expression level of murine IL18 was determined by a commercial ELISA kit. Briefly, BSR cells were infected with rHEP-IL18 or HEP-Flury (MOI=1, 0.1, 0.01, or 0.001) for 48h, and the culture supernatants were harvested for measurement of murine IL18, each value was expressed as mean ±SD from three independent experiments.
Safety of rHEP-IL18 in mice
As shown in Figure 2, mice injected with 1 × 106 FFU of rHEP-IL18 lost less body weight than those injected with the same dose of HEP-Flury. Most of the mice regained their pre-infection body weight by 21 days post-infection (dpi). The data suggest that expression of IL-18 substantially decreased the pathogenicity of the live-attenuated RABV vaccine. No clinical symptoms were observed in all mice, such as abnormal behaviors or any neurological signs.
Figure 2: Pathogenicity of rHEP-IL18 in mice. Groups of 10 ICR mice (6 to 8 weeks old) were infected intracranially with 106 FFU of HEP-Flury, rHEP-IL18 virus or DMEM alone. Their body weights were monitored daily for 21 days. Representative data from all 10 mice in each group and are shown as mean values±SD.
Neurovirulence was also evaluated for the vaccine candidates by i.c. route in suckling mice. One out of ten suckling mice injected via i.c. with 1 × 105 FFU of HEP-Flury died. In contrast, inoculation of suckling mice via i.c. with rHEP-IL18 or DMEM resulted in no clinical signs or lethality. These results suggest that rHEP-IL18 may have an increased safety profile versus HEP-Flury in vivo.
Immunogenicity of rHEP-IL18 in mice
Mice (n = 10) were immunized once via the i.m. route with either 1 × 104 or 1 × 105 FFU per mouse of rHEP-IL18 or HEP-Flury. Mice given DMEM were included as controls. Blood samples were collected at 7, 14, 21, 35, 42 and 56 dpi, and used for the VNA test. As shown in Figure 3A, the level of VNA in mice immunized with 1 × 104 or 1 × 105 FFU rHEP-IL18 (2.26 IU or 2.52 IU, respectively) were higher than that in those induced by immunization with HEP-Flury (< 0.85 IU) at 7 dpi. Significantly higher VNA titers were detected in mice immunized with 1 × 105 FFU of rHEP-IL18 viruses at 14 dpi (56.78 IU) (P < 0.0001) and 21 dpi (41.24 IU) (P < 0.01) than mice immunized with HEP-Flury. Mice immunized with 1 × 104 FFU of rHEP-IL18 induced higher level of VNA than HEP-Flury, but this difference was not statistically significant. VNA titers induced at 35 dpi was similar with that at 21 dpi, however, VNA titers decreased gradually from after 42 dpi to 56 dpi. Significantly higher VNA titers were still detected in mice immunized with 1 × 105 FFU of rHEP-IL18 viruses at 42 dpi (33.91 IU) (P < 0.01) and 56 dpi (27.16 IU) (P < 0.01) than mice immunized with HEP-Flury. No VNA was detected in the DMEM group. Overall, the level of VNA observed is dependent on the vaccination dose.
Figure 3: VNA titers and protection of rHEP-IL18 in mice. (A) Groups of 10 ICR mice (n = 10) were respectively immunized with 1 × 104 or 1 × 105 FFU of rHEP-IL18, HEP-Flury or DMEM by the i.m. route. At days 7, 14, 21, 35, 42 and 56 after immunization, the peripheral blood was collected for VNA test. Titers were normalized to IU using the WHO standard. Data are analyzed by one-way ANOVA and asterisks indicate significant differences among the experimental groups (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001). (B) Then at days 21 after immunization, mice were challenged with 10 × IMLD50 of street rabies virus strain HuNPB3 by the i.m. route and observed daily for 14 days, and survivorship was recorded.
To determine if higher VNA titers correlate with better protection, mice were challenged with 100 x IMLD50 of street rabies virus strain HuNPB3 in the muscle of the forelimb on day 21 after vaccination and observed for development of RABV disease and death for 14 days. As shown in Figure 3B, a higher proportion of survivors were observed among mice immunized with rHEP-IL18 than among those immunized with HEP-Flury. Immunization with 1 × 104 FFU or 1 × 105 FFU of rHEP-IL18 protected 100% of the mice, whereas immunization with 1 × 104 or 1 × 105 FFU of HEP-Flury provided only 40% or 60% protection, respectively. Together, the data indicate that rHEP-IL18 stimulated higher VNA responses and provided better protection compared to HEP-Flury.
Recruitment of B and T cells in vivo
To determine whether the expression of IL-18 results in the recruitment of higher numbers of B and T cells in vivo, flow cytometry was performed to quantify the immune cells in the peripheral blood and lymph nodes (CD3+, CD4+ and CD8+ for T cells and CD19+ and CD40+ for B cells) at 3, 6, and 9 dpi. Each group of mice was immunized once via i.m. with 1 × 105 FFU per mouse of rHEP-IL18 or HEP-Flury. As shown in Figure 4A and 4B, significantly more CD4+ T cells and CD8+ T cells were detected in the blood of mice immunized with rHEP-IL18 than in those immunized with HEP-Flury or mock mice at 3, 6, 9 dpi. Figure 4C, 4D show that significantly more activated B cells (CD40+ and CD19+) were detected in blood and in lymph nodes of mice immunized with rHEP-IL18 than in mice immunized with HEP-Flury or DMEM at 6 and 9 dpi. The results indicate that rHEP-IL18 induced higher levels of T and B cell activation, and that more T and B cells were recruited and/or activated in lymph nodes and peripheral blood.
Figure 4: Recruitment and/or activation of T and B cells in blood and lymph nodes after rHEP-IL18 infection. ICR mice were immunized i.m. with 1 × 105 FFU of rHEP-IL18, HEP-Flury or DMEM. Blood and inguinal lymph nodes were collected from 3 mice per group at 3, 6, and 9 dpi. Single cell suspensions were prepared and stained with antibodies for T (CD3+, CD4+ and CD8+) from the blood (A and B) and B cells (CD19+ and CD40+) from the blood (C) and the lymph nodes (D). Asterisks indicate significant differences among the experimental groups as analyzed by one-way ANOVA (*P < 0.05;**P < 0.01;***P < 0.001;****P < 0.0001).
Cell-mediated immune responses
RABV-specific IFN-γ and IL-4 expression were evaluated in splenocytes by ELISpot assays. As shown in Figure 5A, counts of IFN-γ expressing cells were significantly higher in mice immunized with rHEP-IL18 compared to HEP-Flury (p < 0.01) or DMEM-immunized mice (p < 0.05). As shown in Figure 5B, the rHEP-IL18 induced a higher level of IL-4, but this difference was not statistically significant. The results indicated that rHEP-IL18 elicited a higher Th1 like cellular immune response compared to other groups. The ability of rHEP-IL18 to induce IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells were then measured. As shown in Figure 5C and 5D, rHEP-IL18 elicited higher numbers of IFN-γ- secreting CD4+ and CD8+ T cells, compared with the HEP-Flury and DMEM groups. As shown in Figure 5E and 5F, similar results were observed for IL-4-secreting CD4+ and CD8+ T cells. The data demonstrate that mice immunized with rHEP-IL18 elicited a notably enhanced IFN-γ- or IL-4-secreting CD4+ and CD8 + T cell response.
Figure 5: ELISpot analysis of IFN-γ and IL-4 secretion and ICS assays for antigen-specific CD4+ and CD8+ T cell secretion of IFN-γ and IL-4 in splenocytes. The splenocytes were collected from 3 mice per group at 14 days after immunization, and splenocytes were assayed as described in Material and Methods. The secretion of IFN-γ (A) and IL-4 (B) were measured by a commercial ELISpot kit. Then the splenocytes were stained with mouse anti-CD4, -CD8, -IFN-γ, and -IL-4 monoclonal antibodies. The CD4+ T cells secreting IFN-γ (C) or IL-4 (E), and CD8+ T cells secreting IFN-γ (D) or IL-4 (F) were shown. The data represent the means of subtraction values with SD and were analyzed by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).
IFN-γ, IL-2, IL-4 and IL-10 secretion from splenocytes of immunized mice
Levels of secreted IFN-γ, IL-2, IL-4 and IL-10 from splenocytes were measured using commercial ELISA kits. As shown in Figure 6A and 6D, the levels of IFN-γ and IL-10 secreted in mice immunized with rHEP-IL18 were higher than those detected in the HEP-Flury or mock group. The levels of IL-2 secretion in mice immunized with rHEP-IL18 were significantly higher than those detected in the parent HEP-Flury (P < 0.0001) or DMEM treated mice (P < 0.0001) (Figure 6B). Similarly, the levels of IL-4 secretion in mice immunized with rHEP-IL18 were significantly higher than those detected in the parent HEP-Flury (P < 0.05) or DMEM treated mice (P < 0.01) (Figure 6C). The results indicate that rHEP-IL18 elicited enhanced Th1 and Th2 cellular immune responses.
Figure 6: The quantities of IL-2, IL-4, IL-10 and IFN-γ secreted by splenocytes. Splenocytes were isolated from 3 mice per group at 14 days after immunization, and cell-free supernatants were harvested at 48h after incubation and measured for the levels (pg/mL) of IFN-γ (A), IL-2 (B), IL-4 (C) and IL10 (D) by a commercial ELISA kit in duplicate. Representative data are shown as the mean ± SD of 3 mice per group and were analyzed using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).
Previous studies have shown that the expression of cytokines/chemokines could further attenuate RABV virulence, induce relatively high titers of neutralizing antibodies, and enhance protective immune responses to wild-type RABV challenge [24, 29]. In order to improve the efficacy of RABV vaccines, several cytokines have been trialed as adjuvants to improve immune responses, including MIP-1α, MDC and GM-CSF [29, 30]. In this study, we selected IL-18 on the basis that this cytokines has been widely used as an adjuvant to enhance/regulate innate and adaptive immune responses of many vaccine antigens [31–33].
Our results show that the viral kinetics of rHEP-IL18 (up to 108 FFU) was similar with those of the parent virus HEP-Flury in NA and BSR cells. ICR mice are a kind of out-breed mice, which own strong viability and adaptability, are commonly emplyed for the immunization experiment due to the high repeatability. The pathogenicity and immunogenicity of rHEP-IL18 was then investigated in ICR mice. Although weight loss occurred in mice vaccinated with rHEP-IL18 or HEP-Flury, no overt clinical symptoms, such as abnormal behavior or neurological signs, were observed. However, mice infected with rHEP-IL18 regained their original body weight earlier than mice infected with HEP-Flury, suggesting that IL-18 expression reduced the pathogenicity of the recombinant RABV vaccine.
Our results also showed that rHEP-IL18 induced higher VNA titers and provided better protection than HEP-Flury. Neutralizing antibody is a major component of humoral immunity contributing to protection against RABV infection, and plays an important role in the prevention or reduction of viral spread [34, 35]. Cell-mediated immunity has been proposed to play an important role against RABV infection and may be involved in viral clearance from the CNS [36]. However, it is not known whether IL-18 substantially impacts B cell responses. A previous study has reported that IL-18 has an effect on CD40+ B cell activation [37, 38]. In this study, mice immunized with rHEP-IL18 induced higher titers of VNA at earlier times compared to HEP-Flury at 7 dpi, increasing in titer when tested at 14 and 21 dpi. VNA titers induced at 35 dpi was similar with that at 21 dpi, and decreased gradually from 42 dpi. Furthermore, our data showed that IL-18 stimulated the maturation, activation, and/or recruitment of more CD19+ CD40+ B cells in lymph nodes and blood among immunized mice than HEP-Flury at 3, 6 and 9 dpi, which may contribute to the higher VNA induced than the parental virus.
IL-18 has a potent ability to produce IFN-γ, which activates immune cells either independently or with IL-12 [39]. The Th1 immune response is activated by IL-2 and IFN-γ [40, 41], whereas the Th2 immune response is activated by IL-4 and IL-10 [42]. Our data showed that rHEP-IL18 enhanced cytokines secretion of IFN-γ and IL-10, and had a significant effect on IL-2 and IL-4 cytokines secretion, indicating that rHEP-IL18 mediated/boosted immune responses via both the Th1 and Th2 pathways. Also, rHEP-IL18 induced a stronger and faster CD4+ and CD8+ T cell response in immunized mice in vivo than HEP-Flury, with higher numbers of peripheral CD4+ and CD8+ cells observed at 3, 6 and 9 dpi. The combination of the humoural and cell-mediated immune response data suggests a stronger adaptive immune response contributed to enhanced protection in rHEP-IL18-vaccinated mice.
Overall, these results demonstrated that IL-18 possesses strong adjuvant activity and that rHEP-IL18 enhanced humoral and cellular immune responses leading to enhanced protection. Therefore, the rHEP-IL18 is an improvement on the HEP-Flury vaccine and should be further characterized in a higher-order animal model.
Animal studies were carried out in strict accordance with prior approval from the Animal Welfare and Ethics Committee of School of Public Health, Shandong University (permit number SCXK-20150015). The environment and housing facilities used in this experiment satisfies the guidelines put forth by the National Standards of Laboratory Animal-Requirements of Environment and Housing Facilities (GB 14925-2001) of China. All efforts were made to minimize animal suffering.
Cells, viruses, antibodies, and animals
BSR cells, a cloned cell line derived from BHK-21 cells, and mouse neuroblastoma (NA) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Invitrogen, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA). The RABV HEP-Flury reverse genetics system is provided by Professor Xiaofeng Guo (South China Agricultural University). Standard attack rabies virus strain CVS-11 and street rabies virus strain HuNPB3 were propagated in NA cells. Fluorescein isothiocyanate (FITC)-conjugated antibody against the RABV N protein was purchased commercially from Fujirebio Diagnostics, Inc. (Malvern, PA, USA). Anti-IL-18 monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Antibodies used for flow cytometry analysis, such as anti-CD3, anti-CD4, anti-CD8, anti-CD19 and anti-CD40 were purchased commercially (BD Biosciences, USA). Female ICR mice (6-8 weeks old) were purchased from the Shandong University Laboratory Animal Center (Jinan, China).
Construction of recombinant RABV cDNA clones
The vector HEP-Flury was digested with BsiWI and NheI between the G and RNA-dependent RNA polymerase (L) genes. Murine IL-18 was amplified from the mouse genome and inserted between the G and L genes to obtain HEP-Flury-IL18.
Rescue of rRABV HEP-IL18
rHEP-IL18 was rescued as described previously [43, 44]. Briefly, 1 × 106 BSR cells per well were grown overnight to 60%–80% confluence in six-well plates (Corning, Steuben County, NY, USA) in DMEM supplemented with 10% FBS. The cells were transfected with 2.0 μg of the full-length clone, 0.5 μg of N helper plasmid, 0.25 μg of P helper plasmid, 0.1 μg of L helper plasmid and 0.15 μg of G helper plasmid using the SuperFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer instructions. After incubation at 37°C for 4 days, the culture medium was harvested and the cells were tested for rescued virus using FITC-conjugated anti-RABV N antibody (Centocor, Malvern, PA, USA). Fluorescence in the cells was measured with a fluorescence microscope. The rescued virus rHEP-IL18 strain was inoculated in NA cells and BSR cells for virus stock preparation and further experiments.
Virus titration
Virus titration was performed using the direct fluorescent antibody assay in NA cells. In a 96-well plate, NA cells were inoculated with serial 10-fold dilutions of virus and incubated at 37 °C for 48 h. The culture supernatant was removed, and the cells were fixed with ice-cold 80% acetone for 30 min. The cells were washed twice with PBS and then stained with FITC-conjugated anti-RABV N antibody at 37 °C. Antigen-positive foci were counted under a fluorescence microscope and virus titer was calculated as fluorescent focus units per milliliter (FFU/mL). All titrations were carried out in quadruplicate.
Multistep growth assays
Monolayer cultures of NA or BSR cell grown in a T25 cell culture flask were infected with HEP-Flury or rHEP-IL18 at an MOI of 0.01. After inoculation for 1h at 37 °C, the supernatant were removed and then the cells were washed twice with PBS (pH7.4). The NA or BSR cells were supplied with fresh growth medium containing 2% FBS and incubated in a 5% CO2 incubator at 37 °C. Samples of culture medium were harvested from the NA or BSR cells at 24, 48, 72, 96, 120 and 144 h post-infection for virus titration. Virus titer was determined in BSR cells using the direct fluorescent antibody as previously described.
BSR cells were infected with different viruses at an MOI of 0.01 and incubated at 37 °C for 48 h and lysed with RIPA buffer (Thermo Scientific, USA). The lysates were centrifuged at 12,000 ×g for 15min and the supernatants were mixed with 5 × loading Buffer. IL-18 expression was analyzed by SDS-PAGE and transferred onto a fluoride (PVDF) membrane (Immobilin-P, Millipore, USA) for Western blotting with anti-IL-18 monoclonal antibody (1:500), followed by detection with HRP-conjugated goat anti-mouse IgG polyclonal antibody (1:20,000) and enhanced chemiluminescence.
Safety studies
To determine whether rHEP-IL18 vaccination is safe in mice, 3 groups of 10 female ICR mice (6 to 8 weeks old) were administered 1 × 106 FFU of rHEP-IL18, HEP-Flury or DMEM via the intracerebral (i.c.) route. Body weights were monitored daily for 21 days. Data were obtained from 5 mice in each group and presented as the mean value ± standard deviation (SD).
For neurovirulence analyses, 3 groups of 10 ICR suckling mice (5-day-old) were administered 1 × 106 FFU of rHEP-IL18, HEP-Flury or DMEM via the i.c. route. Suckling mice were monitored daily for encephalitis and the numbers of surviving mice were recorded daily.
Immunogenicity and protection studies
Female ICR mice (6-8 weeks old) were randomly divided into 5 groups and vaccinated intramuscularly (i.m.) in the hind legs with HEP-Flury or rHEP-IL18 at a concentration of 104 or 105 FFU. A control group was mock-vaccinated with DMEM at the same time points. At 21 days after immunization, mice were challenged i.m. with 100 × median intramuscular mouse lethal dose (IMLD50) of street rabies virus strain HuNPB3 in the muscle of the forelimb, and survival/body weights were observed daily for 21 days. During the observation period, any mice that developed clinical signs of rabies were humanely euthanized by cervical dislocation under isoflurane anesthesia.
Fluorescent antibody virus neutralization tests (FAVN)
Blood was collected from mice 7, 14, 21, 35, 42 and 56 days after immunization by retro-orbital plexus puncture for measurement of VNA using a fluorescent antibody virus neutralization (FAVN) test as described previously [45]. Titers were calculated according to the Spearman and Kärber method [46].
To investigate the effects of IL-18 expression on the recruitment of T and B cells in the peripheral blood and in lymph nodes, flow cytometry was performed using a LSR-II flow cytometer (BD Bioscience). Briefly, 10 female ICR mice (6-8 weeks) per group were inoculated i.m. with 1 × 105 FFU of HEP-Flury, rHEP-IL18 or with medium alone. At days 3, 6 and 9 post-infection, the single cell suspension of peripheral blood or inguinal lymph nodes was harvested and stained with antibodies to T cell- (CD3+, CD4+, and CD8+) or B cell- (CD19+ and CD40+) markers (BD Biosciences Pharmingen, USA) for 30 min at 4 °C. A minimum of 20,000 events were counted.
ELISpot assays
Splenocytes from vaccinated mice were isolated at 14 days after immunization and stimulated with inactivated HuNPB3 at a concentration of 10 μg/mL for 36 h at 37 °C. Secreted IFN-γ or IL-4 was quantified by the mouse IFN-γ/IL-4 ELISPOT kit (Mabtech AB, Stockholm, Sweden) according to manufacturer instructions. Spot forming cells (SFCs) were counted using an automated ELISpot reader (AID ELISPOT reader-iSpot, AID GmbH, GER).
Intracellular cytokine staining (ICS)
Splenocytes were collected from 3 mice from each group at 14 days after immunization and were stimulated with inactivated HuNPB3 (10 μg/mL) with a protein transport inhibitor (BD Biosciences, Franklin, TN, USA). At 6 h post-stimulation, single cell suspensions were stained with anti-CD4 and anti-CD8 monoclonal antibodies (BD Biosciences, Franklin, TN, USA) for 30 min at 4 °C, as well as anti-IFN-γ and anti-IL-4 monoclonal antibodies (BD Biosciences, Franklin, TN, USA) for 30 min at 4 °C. All labeled cells were washed twice with PBS and 20,000 cells were analyzed in a LSR-II flow cytometer (BD Bioscience, Franklin, TN, USA).
IL-2, IL-4, IL-10 and IFN-γ expression analysis using ELISA
Splenocytes were harvested from 3 mice of each group at 14 days after vaccination. The splenocytes (2 × 106 cells/mL) were suspended in RPMI 1640 medium containing 10% FBS, and stimulated with inactivated HuNPB3 (10 μg/mL) at 37 °C for 48 h. The culture supernatants were then harvested and determined using an ELISA kit (Mouse IL-2, IL-4, IL-10 and IFN-γ ELISA kit, Mabtech AB). All assays were performed according to manufacturer instructions.
The data were expressed as the mean ±SD and analyzed using GraphPad Prism 6 to determine statistically significant differences in the generated data by one-way analysis of variance (ANOVA). P < 0.05 was considered a statistically significant difference. *P <0.05;**P < 0.01; ***P < 0.001;****P < 0.0001.
This work was supported by The National Key Research and Development Program of China (2016YFD0501003), The National Natural Sciences Foundation of China (31402173), Shandong Provincial Natural Science Foundation (ZR2016CQ18) and the China Postdoctoral Science Foundation funded project (2015M580592, 2016T90637). G.W. is the recipient of a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (CIHR) and the President's International Fellowship Initiative from the Chinese Academy of Sciences (CAS).
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14. Blancou J, Artois M, Brochier B, Thomas I, Pastoret PP, Desmettre P, Languet B, Kieny MP. Safety and efficacy of an antirabies vaccine consisting of recombinant vaccinia-rabies virus administered orally to the fox, dog and cat. [Article in French]. Ann Rech Vet. 1989; 20: 195-204.
15. Brochier B, Kieny MP, Costy F, Coppens P, Bauduin B, Lecocq JP, Languet B, Chappuis G, Desmettre P, Afiademanyo K. Large-scale eradication of rabies using recombinant vaccinia-rabies vaccine. Nature. 1991; 354: 520-2. https://doi.org/10.1038/354520a0.
16. Flamand A, Coulon P, Lafay F, Tuffereau C. Avirulent mutants of rabies virus and their use as live vaccine. Trends Microbiol. 1993; 1: 317-20.
17. Hanlon CA, Niezgoda M, Hamir AN, Schumacher C, Koprowski H, Rupprecht CE. First North American field release of a vaccinia-rabies glycoprotein recombinant virus. J Wildl Dis. 1998; 34: 228-39. https://doi.org/10.7589/0090-3558-34.2.228.
18. Cliquet F, Aubert M. Elimination of terrestrial rabies in Western European countries. Dev Biol (Basel). 2004; 119: 185-204.
19. Niin E, Barrat J, Kristian M, Demerson JM, Cliquet F. First oral vaccination of wildlife against rabies in Estonia. Dev Biol (Basel). 2006; 125: 145-7.
20. Roscoe DE, Holste WC, Sorhage FE, Campbell C, Niezgoda M, Buchannan R, Diehl D, Niu HS, Rupprecht CE. Efficacy of an oral vaccinia-rabies glycoprotein recombinant vaccine in controlling epidemic raccoon rabies in New Jersey. J Wildl Dis. 1998; 34: 752-63. https://doi.org/10.7589/0090-3558-34.4.752.
21. Robbins AH, Borden MD, Windmiller BS, Niezgoda M, Marcus LC, O'Brien SM, Kreindel SM, McGuill MW, DeMaria A Jr, Rupprecht CE, Rowell S. Prevention of the spread of rabies to wildlife by oral vaccination of raccoons in Massachusetts. J Am Vet Med Assoc. 1998; 213: 1407-12.
22. Phares TW, Kean RB, Mikheeva T, Hooper DC. Regional differences in blood-brain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system. J Immunol. 2006; 176: 7666-75.
23. Roy A, Hooper DC. Immune evasion by rabies viruses through the maintenance of blood-brain barrier integrity. J Neurovirol. 2008; 14: 401-11. https://doi.org/10.1080/13550280802235924.
24. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature. 1995; 378: 88-91. https://doi.org/10.1038/378088a0.
25. Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S, et al. Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol. 1996; 156: 4274-9.
26. Zhang X, Li G, Gao L, Mu L, Zhang L, Cong Y, Ding Z. Positive inductive effect of IL-18 on virus-specific immune responses induced by PRRSV-GP5 DNA vaccine in swine. Res Vet Sci. 2013; 94: 346-53. https://doi.org/10.1016/j.rvsc.2012.09.011.
27. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev. 2001; 12: 53-72.
28. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol. 2001; 19: 423-74. https://doi.org/10.1146/annurev.immunol.19.1.423.
29. Wen Y, Wang H, Wu H, Yang F, Tripp RA, Hogan RJ, Fu ZF. Rabies virus expressing dendritic cell-activating molecules enhances the innate and adaptive immune response to vaccination. J Virol. 2011; 85: 1634-44.
30. Zhou M, Zhang G, Ren G, Gnanadurai CW, Li Z, Chai Q, Yang Y, Leyson CM, Wu W, Cui M, Fu ZF. Recombinant rabies viruses expressing GM-CSF or flagellin are effective vaccines for both intramuscular and oral immunizations. PLoS One. 2013; 8: e63384. https://doi.org/10.1371/journal.pone.0063384.
31. Noll A, AutenriethIb. Immunity against Yersinia enterocolitica by vaccination with Yersinia HSP60 immunostimulating complexes or Yersinia HSP60 plus interleukin-12. Infect Immun. 1996; 64: 2955-61.
32. Schneider K, Puehler F, Baeuerle D, Elvers S, Staeheli P, Kaspers B, Weining KC. cDNA cloning of biologically active chicken interleukin-18. J Interferon Cytokine Res. 2000; 20: 879-83. https://doi.org/10.1089/10799900050163244.
33. Gobel TW, Schneider K, Schaerer B, Mejri I, Puehler F, Weigend S, Staeheli P, Kaspers B. IL-18 stimulates the proliferation and IFN-gamma release of CD4+ T cells in the chicken: conservation of a Th1-like system in a nonmammalian species. J Immunol. 2003; 171: 1809-15.
34. Li J, Ertel A, Portocarrero C, Barkhouse DA, Dietzschold B, Hooper DC, Faber M. Postexposure treatment with the live-attenuated rabies virus (RV) vaccine TriGAS triggers the clearance of wild-type RV from the Central Nervous System (CNS) through the rapid induction of genes relevant to adaptive immunity in CNS tissues. J Virol. 2012; 86: 3200-10. https://doi.org/10.1128/JVI.06699-11.
35. Liao PH, Yang HH, Chou PT, Wang MH, Chu PC, Liu HL, Chen LK. Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats. J Biomed Sci. 2012; 19: 61. https://doi.org/10.1186/1423-0127-19-61.
36. Johnson N, Cunningham AF, Fooks AR. The immune response to rabies virus infection and vaccination. Vaccine. 2010; 28: 3896-901. https://doi.org/10.1016/j.vaccine.2010.03.039.
37. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both TH1 and TH2 responses. Annual Review of Immunology. 2001; 19: 423-74. https://doi.org/10.1146/annurev.immunol.19.1.423.
38. Yoshimoto T, Okamura H, Tagawa YI, Iwakura Y, Nakanishi K. Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon-gamma production from activated B cells. Proc Natl Acad Sci U S A. 1997; 94: 3948-53.
39. Barbulescu K, Becker C, Schlaak JF, Schmitt E, Meyer zum Buschenfelde KH, Neurath MF. IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN-gamma promoter in primary CD4+ T lymphocytes. J Immunol. 1998; 160: 3642-7.
40. Swain SL, McKinstry KK, Strutt TM. Expanding roles for CD4(+) T cells in immunity to viruses. Nat Rev Immunol. 2012; 12: 136-48. https://doi.org/10.1038/nri3152.
41. Sant AJ, McMichael A. Revealing the role of CD4(+) T cells in viral immunity. J Exp Med. 2012; 209: 1391-5. https://doi.org/10.1084/jem.20121517.
42. Nair S, Bayer W, Ploquin MJ, Kassiotis G, Hasenkrug KJ, Dittmer U. Distinct roles of CD4+ T cell subpopulations in retroviral immunity: lessons from the Friend virus mouse model. Retrovirology. 2011; 8: 76. https://doi.org/10.1186/1742-4690-8-76.
43. Inoue K, Shoji Y, Kurane I, Iijima T, Sakai T, Morimoto K. An improved method for recovering rabies virus from cloned cDNA. J Virol Methods. 2003; 107: 229-36.
44. Takayama-Ito M, Inoue K, Shoji Y, Inoue S, Iijima T, Sakai T, Kurane I, Morimoto K. A highly attenuated rabies virus HEP-Flury strain reverts to virulent by single amino acid substitution to arginine at position 333 in glycoprotein. Virus Res. 2006; 119: 208-15. https://doi.org/10.1016/j.virusres.2006.01.014.
45. Cliquet F, Aubert M, Sagne L. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabies-neutralising antibody. J Immunol Methods. 1998; 212: 79-87.
46. Kasermann F, Kempf C. Photodynamic inactivation of enveloped viruses by buckminsterfullerene. Antiviral Res. 1997; 34: 65-70.


text:
Q: jQuery, Set Item In One Page, Display It In Another I have an HTML page with a textbox named "workArea":
<button id="saveWork">Save Your Work</button>

<textarea id="workArea" cols="80" rows="40">
User edits text here.
</textarea>

I want to use jQuery to, when user clicks button "saveWork":

*

*change the button to a confirmation that user's work is saved (is working)

*save the value inside textbox "workArea" to local storage under 'workData' (not working)

var saveConfirm = "Your work has been saved!";
$("#saveWork").click(function() {
 $('#saveWork').replaceWith(document.createTextNode(saveConfirm)).show();

 localStorage. setItem ('workData',document.getElementById('workArea').value);

});

Then, on a new HTML page, display the saved value inside "workArea" in another textbox for the user to continue working on.
Which I figure is something to do with putting this inside a script tag on the new HTML page...

$(document).ready(function() { localStorage.getItem('workData'); });

I am unable to get the new HTML page to display anything.
Where am I going wrong?
Thanks!

A: Here is a Demo Fiddle
$('#Output').val(localStorage.getItem('workData'));

Refresh the page after you Click on 'Save your work', you will get the output in the small text area.

redpajama_set_name: RedPajamaStackExchange


text:
poster="http://v.politico.com/images/1155968404/201512/2883/1155968404_4665620796001_151217-Reid-00-00-13-00-Still001.jpg?pubId=1155968404"
Bad blood: Reid-McConnell relationship hits new low
The Senate leaders met for a rare one-on-one meeting in late November, and it couldn't have gone much worse.
By BURGESS EVERETT
Harry Reid and Mitch McConnell sat down for a rare face-to-face meeting on the last day of November. The agenda was straightforward — plotting out the legislative agenda for the end of the year — but what transpired has pushed the relationship between the veteran Senate leaders, never warm and fuzzy, to a new low of bitterness and distrust.
Reid left the meeting seething with rage, associates of the Democratic leader said, after McConnell told Reid that legislative language to help 9/11 responders and challenge a boycott against Israeli goods would be left out of a must-pass transportation bill. Within a day, Reid's allies were going public to portray McConnell as using 9/11 heroes as negotiating chips, and later that week Reid said it was solely McConnell who blocked the Israel language.
Then it was McConnell's turn to vent: Not only did he deny Reid's charges publicly, but nearly a week later, McConnell (R-Ky.) was still privately grousing that Reid used the closed-door meeting to attack McConnell politically, sources said.
As House Speaker Paul Ryan (R-Wis.) and Minority Leader Nancy Pelosi (D-Calif.) have spent the past few weeks chatting on the phone and sharing a steak dinner like old high school pals, McConnell and Reid can hardly stand the sight of each other. They've met just once in December, even as their parties have been furiously negotiating the year-end spending and tax bills. Though Reid and McConnell have communicated through aides and occasionally with each other on the floor, they've done so with gritted teeth.
Reid, McConnell insist they like each other
"It's just not the same relationship. … Maybe it's my fault," Reid said in a reflective moment during an interview last month. "It's not as if we're bitching at each other all the time. Our staffs get along. It's just that we don't get together very much."
Together, the two legislative tacticians delivered major accomplishments this year: a budget deal, entitlement fixes and a big transportation package. But it's clear through interviews with associates, senators and the leaders themselves they may never be able to let go of their argument, which has lasted for years, over which of them broke the Senate.
McConnell was just beginning his monthlong victory lap on Dec. 2, two days after Reid was informed his Israel and 9/11 priorities would be dropped by McConnell. The topic of McConnell's speech that day: The Senate was "back to work" and now passing "bills no one ever thought Washington could do."
Often, the two leaders just talk past each other in their daily floor speeches, but on this day Reid had had enough. McConnell's Senate, the minority leader declared, has been "the most unproductive Senate in the history of the country."
A week later, Reid was whacked with three Pinocchios by The Washington Post Fact Checker, a ruling that rattled around the leadership suites but was largely ignored otherwise. The constant litigation of the Senate's productivity may not resonate much outside the Capitol, but McConnell was ebullient.
"By any objective standard, this has been a very successful first year. [The Post] gave Harry three Pinocchios," McConnell said in an interview, flashing a rare grin.
Leaders confident rank-and-file will back huge budget deal
By JAKE SHERMAN, LAUREN FRENCH and ANNA PALMER
To McConnell and the retiring Reid, the story of which leader is to blame for turning the Senate into a gridlocked legislative wasteland most of the time often seems as important as the next accomplishment.
Reid thinks McConnell is essentially just passing bills that he blocked a year ago only to take credit for them now. When asked about McConnell's victory lap, which has included numerous national interviews focused almost solely on McConnell's productive first year, Reid at first tried to be diplomatic.
But a year after being demoted from the majority, Reid still can't get over what he views as McConnell's obstruction during the preceding Congress.
"How many filibusters has he had to overcome? I had hundreds of them," Reid said as he ambled to his SUV on Tuesday night. "The reason he's done well is we've helped him."
The incessant sparring over who ran the Senate better, how many filibusters were mounted, who got more amendment votes and which leader has been more cooperative is exhausting to many senators. Some say they rarely tune in anymore to hear the latest from Reid about how terrible Republicans are and how poorly McConnell runs the Senate, or to hear McConnell brag about his leadership.
"I probably don't want this in print, but I'll say it anyway. I don't listen to much of their opening remarks," said Sen. Jon Tester (D-Mont.). "It's kind of wasted energy. We've got work to do here."
On paper, Reid and McConnell have much in common: They were party whips together, then leaders together since 2007. The two men are both in their 70s and have spent decades in the Senate. Each knows the rules, procedures and rhythms of the Senate better than anyone else in the Capitol.
Even their harshest critics concede that Reid and McConnell both know how to seal a deal.
But besides their shared love for the Washington Nationals, they couldn't be more different: Reid is a spontaneous speaker who revels in a good insult and has never taken to Capitol Hill's country club culture. His upbringing in Searchlight, Nevada, can seem otherworldly. Aides once asked him how he brushed his teeth growing up without plumbing; he called his brother to learn that, as a young boy, his family didn't even own toothbrushes.
McConnell is a fixture at Washington meetings with party honchos and at fundraisers. He's also beyond reserved, leaving his closest deputies guessing about his next move. Reid will fly off the handle in an interview if given the right question; McConnell is perfectly happy to keep declining comment until a reporter moves on to something that he wants to talk about.
"We just have different styles, you know. I don't think it's productive to throw bombshells around," McConnell said this month. "That's just not my style. I like Harry personally, we spend a lot of time together. I think you would agree, I'm more restrained."
Privately, people who know both men well say that things have always been rocky, but the relationship took a sharp turn south after Reid changed the Senate's rules to confirm President Barack Obama's nominees more easily. That move was in response to McConnell's constant use of delaying tactics.
Each blames the other for the escalation of the nomination wars: Republicans say Reid started it with George W. Bush's picks, while McConnell rarely let a nominee slip by last year without a procedural vote.
But their meeting on Nov. 30, marked a new nadir. Reid allies say McConnell had left the Democratic leader with the impression that his priorities were safe, only to pull them out from under Reid after it was too late to save them.
"That was something that Harry just couldn't understand," said Senate Minority Whip Dick Durbin (D-Ill.).
Sen. John McCain (R-Ariz.) said there's another factor souring their relationship: Reid's brutal injury in January that occurred when an exercise band snapped and sent him flying, leaving him mostly blind in one eye.
Ryan calls Pelosi on spending deal
By JOHN BRESNAHAN and JAKE SHERMAN
"It seems to me that Harry has, since his accident, become a bit more angry about things," McCain said in an interview. He added, "I'm not a psychologist or anything like that, but since his accident there's been an edge that I have not seen before … and it's authenticated when he makes his comments."
Asked to respond to McCain, Reid spokeswoman Kristen Orthman said: "Sen. Reid has great respect and affection for all of his colleagues, including Sen. McCain. If Republicans are frustrated, it's probably because he tends to get the better of them in negotiations."
Among Republicans, Reid's name spells Senate dysfunction. Last year, the GOP, with some success, built their campaign to retake the Senate around the slogan, "Fire Harry Reid."
While that may have worked, the GOP was still left with Reid as the Democratic leader. And Reid showed this year he can still whip his caucus to shut down bills and that the softspoken onetime boxer still has one of the loudest microphones in Washington to cast the GOP as being out of step with voters.
Republicans are hoping that Reid's heir apparent, New York Sen. Chuck Schumer, will help heal the scars left by nine years of battles between McConnell and Reid.
"I actually think things will improve," said Senate Majority Whip John Cornyn (R-Texas). "Sen. Schumer is certainly capable of spinning himself up and being a tough partisan. But I think there's also a practical side to him, too."
Schumer declined to comment for this story, other than to take Reid's side.
"Sen. McConnell wants to see the Senate work," Schumer said. "But the good news for us is, to make it work, he has to do basically our agenda."

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Prolog Program to Find Square of Natural Numbers My code below is meant to generate the square of natural numbers in order
(i.e sq(X). -> X=0; X=1; X=4; X=9; X=16; ...)
nat(0).
nat(X) :- nat(Y), Z is Y+1, X is Z*Z.

but the answer I am getting is:
1
0 ?- nat(X).

X = 0 ;

X = 1 ;

X = 4 ;

X = 25 ;

X = 676

Should be a quick fix, but I've spent longer on this than I'd like to say. Any help is greatly appreciated!

A: your nat/1 really seems to return a different sequence. Should be
nat(0).
nat(X) :- nat(Y), X is Y+1.

and then, a different predicate for square
sq(X) :- % call nat/1, square it...

please complete the code


text:
Smoking to be banned at all London bus shelters
March 11, 2015 - Martin Hoscik@martinhoscik
Image: Lucian Milasan / Shutterstock.com
A commitment from the Mayor to install 'no smoking' signs at almost 10,000 bus shelters across London has been welcomed by Liberal Democrats on the London Assembly.
Signs are already in place at 2,700 shelters but Boris Johnson has now instructed Transport for London to fit them at the remaining 9,800 by the end of April.
Caroline Pidgeon, the Liberal Democrat leader at City Hall, said: "Whatever one thinks about smoking no one, and especially children, should be forced to inhale cigarette smoke simply because they are waiting for a bus.
"This long overdue decision to make all of London's bus shelters no smoking is good news for the lungs of London's children."
Stephen Aselford says
While I welcome the decision, How will this be enforced across London
Mariatu Kamara says
Is a great development for we Londoners that are using the bus shelters, because if somebody is smoking
at the bus shelter and you are non smoker you don't want to inhale the smoke you have to
move out of the shelter and wait outside even if it's raining. so it's really good for our health.


Can someone please advise which file to edit to restrict the size of a custom field?
How can we reduce the size of a custom field of type Text Field (Multi Line).
We need to restrict the size to 30 characters.
If I am not wrong, we need to add below content in edit-textarea.vm file right?
If you don't mind can you give the attribute and in which line to add it?
Er, you need to understand the html changes you are making for yourself.


Spanish style hacienda with large arches and doorways. This house was built in 1949, with major repairs in 2014 retaining its splendid architecture of large living rooms and spacious bed rooms with sophisticated décor, tasteful mixing of the most dissimilar artifacts from the Cuban golden age with European and modern style furniture. This makes Cañaveral House one of the most beautiful places in Havana, ideal for staying at on your visit to Cuba.
Located in the Playa municipality just a few minutes (8min) from Vedado town center and 12min from the legendary Habana Vieja, nearby the International cosmetic surgery clinic and other pathology "Cira Garcia" as well as the famed Tropicana cabaret.
With three beautiful bedrooms all with private bathrooms, this house is ideal for families or groups of friends.


Jadroagent dd -International Shipping and Freight agency, was founded in 1947. and was incorporated as limited company in 2004. Company presently employees about 190 people. Beside Rijeka where we have our head office, we hold own offices in all Croatian ports: Pula , Rasa , Zadar, Sibenik, Split, Ploce, Dubrovnik and in Zagreb.
Main activities are ship agency, forwarding and container operations, cargo booking and chartering for both dry cargo and liquid cargoes, bunker brooking, port agency and travel agency. We are also Lloyd\'s agent and P&I Correspondents for major clubs worldwide. Company is one of the biggest shipping and freight agency in the area with well trained and experienced staff.


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text:
Here we tap into dates from M.C. Eschers life and work, jumping through time but always in the now. All year round you can enjoy background stories, anecdotes and trivia about this fascinating artist.
Escher in 2021
2021 has come to an end and it has been another special and unforgettable year. We look back on all the wonderful things we have been able to organise this year. No fewer than four temporary exhibitions have been held in the museum, in addition to the permanent presentation of Escher's art!
All of the wonderful images about our exhibitions, activities and the in-depth articles on our website can be seen in this special end-of-year animation. We thank everyone for their attention and support over the past year and hope to inspire you again in 2022 with our stories and artworks!
In November 1929, Escher produced a print that for once was not the direct result of a journey he had made that spring. From 1925 to 1936, he followed a fixed pattern of travelling through Italy in the spring, to the Abruzzi, Sicily, Calabria or the Amalfi Coast. In the first few years, places around his home town of Rome or in the nearby province of Viterbo were added. He also travelled to Corsica and Spain. In the autumn and winter following these trips, he fleshed out his sketches and photos into prints. But in May 1926, things were different.
Hein 's-Gravesande
On 2 July 1965, journalist, poet, critic and essayist G.H. 's-Gravesande, known by his nickname Hein, died. Although Escher and Hein had been friends for over 30 years, the artist did not attend the cremation. He was engrossed in working on the print Knots, a subject in which he had become completely absorbed. This would hardly have surprised 's-Gravesande. He published several articles and a booklet on the man whom he also greatly admired as an artist. Hein 's-Gravesande was one of the first critics to pay serious attention to the work of M.C. Escher, and the graphic artist owes much to him.
Graphic arts friends
M.C. Escher is undoubtedly the most famous graphic artist in the Netherlands. But he was certainly not the only one, as evidenced by our exhibition Graphic Grandeur: Escher and his Contemporaries. Escher was in contact with fellow graphic artists and in a number of cases this also led to joint exhibitions. He was trained as a graphic artist and was indeed an artist, but he struggled with that label all his life. He situated himself more in the tradition of artist-as-craftsman. To be able to make graphic art, it was first and foremost important to have a solid mastery of the necessary techniques. That was true for Escher himself as well as for colleagues. He always appreciated meeting graphic artists who were also masters of their craft. He liked to surround himself with these craftsmen.
An ode to Emma
The final print in Graphic Grandeur: Escher and his Contemporaries is an ode not to Escher, but to the first royal resident of this palace: Queen Emma, the Queen Mother. In this 1897 lithograph by Jan Toorop (1858-1928) we see Emma and her daughter Wilhelmina on a visit to Gouda. The print features all kinds of objects associated with industry in Gouda at the time, such as pipes and candles. The litho is made in Toorop's distinctive Art Nouveau style, recognisable by its strong contours with graceful lines and exuberant decorative elements. This style was also popularly called the Salad Oil Style, in response to the iconic decorative poster that Toorop made for the Nederlandsche Oliefabriek (Dutch Oil Factory) in Delft.
In search of the experiment
The history of printmaking goes back for centuries. So it is no wonder that a great range of printmaking techniques have been developed over time. From woodcut to copper engraving and from mezzotint to screen printing. Moreover, many graphic artists have successfully added their own personal twist to this ancient craft. By experimenting they paved the way for technological improvements, but also for new modes of artistic expression. Experiments formed part of printmaking from the very beginning. In the 17th century, for example, the Dutch artist Hercules Seghers inked his etchings with oil paint, bringing color to the black-and-white world of etching.
Mortality immortalised: Julie de Graag & M.C. Escher
Memento mori: this old Latin phrase reminds people that we will all die some day. This saying is the gloomy subject of the simple yet direct woodcut of Julie de Graag (1877-1924). De Graag was a talented graphic artist and her work was highly stylised. Influenced by sculptor Joseph Mendes da Costa and De Stijl's Bart van der Leck, she increasingly omitted details, as her linework grew simpler and more direct.
Richard Roland Holst
The term homo universalis, meaning universal man, aka polymath, was coined in the Renaissance by the writer, philosopher and musician Leon Battista Alberti (1404-1472). Leonardo da Vinci is often seen as the quintessential polymath. In his case, this referred to his mastery of the complete spectrum of sciences. Greek philosopher Aristotle (384-332 BC) is considered to be the first homo universalis. The term is at times applied incorrectly, but Richard Roland Holst (1868-1938) definitely qualifies. In the database of the RKD, the Netherlands Institute for Art History, he is described as an author, sculptor, scene-painter, Academy director, etcher, glass painter, professor, illustrator, lithographer, furniture designer, designer, painter, draftsman, maker of woodcuts and muralist. A universal man of the arts, in other words.
A Parade of Portraits
The tradition of portraiture goes back centuries. An entire room is devoted to this subject in the exhibition Graphic Grandeur: Escher and his Contemporaries. You are not the only one looking here in this gallery. Lots of eyes are looking back at you, too: from Beethoven to a stylised dog, and from Escher's wife Jetta to a Dutch General with one eye. As soon as the exhibition opens to the public (hopefully as soon as possible!), you can look and be seen here.
Samuel Jessurun de Mesquita
Samuel Jessurun de Mesquita was a gifted artist, painter and printmaker with an idiosyncratic signature who occupies a special place in the canon of art. But above all he is the discoverer of M.C. Escher, the man who made the architecture student choose the profession that would make him world-famous. The sorcerer's apprentice was to outshine his discoverer and things slowly grew quieter around De Mesquita. On 31 January 1944 he was arrested by the Nazis. He died shortly afterwards in Auschwitz concentration camp. Escher was devastated and the death of his teacher made a deep impression on him. Nowadays the name of Samuel Jessurun de Mesquita is often directly linked to that of Escher, but there is still plenty to say about the teacher.
More Escher today
Oscar Reutersvärd
With Belvedere, Waterfall and Ascending and Descending, M.C. Escher created three iconic prints based on impossible figures: a cube, a triangle and a staircase. He invented the one for Belvedere himself, but the impossible triangle and the infinite staircase were presented to him by the British mathematicians Lionel and Roger…
Between March and June 1931, Escher created his Emblemata, a series of small woodcuts that were accompanied by a motto in Latin and a poem in Dutch. The mottoes and poems were written by art historian G.J. Hoogewerff, director of the Dutch Historical Institute in Rome and a friend to…
2020 has passed. A year no one will ever forget. But even in this crazy year, we brought you many stories about the life and work of M.C. Escher. All the images we used are collected in this video. We thank everyone for your attention this year and onwards to…


text:
import { combineFrom } from 'inferno-shared';
import { EMPTY_OBJ, VNode } from 'inferno';
import { VNodeFlags } from 'inferno-vnode-flags';

const rxUnescaped = new RegExp(/["'&<>]/);

export function escapeText(text: string): string {
  /* Much faster when there is no unescaped characters */
  if (!rxUnescaped.test(text)) {
    return text;
  }

  let result = '';
  let escape = '';
  let start = 0;
  let i;
  for (i = 0; i < text.length; ++i) {
    switch (text.charCodeAt(i)) {
      case 34: // "
        escape = '"';
        break;
      case 39: // '
        escape = ''';
        break;
      case 38: // &
        escape = '&';
        break;
      case 60: // <
        escape = '<';
        break;
      case 62: // >
        escape = '>';
        break;
      default:
        continue;
    }
    if (i > start) {
      result += text.slice(start, i);
    }
    result += escape;
    start = i + 1;
  }
  return result + text.slice(start, i);
}

const uppercasePattern = /[A-Z]/g;

const CssPropCache = {};

export function getCssPropertyName(str): string {
  if (CssPropCache[str] !== void 0) {
    return CssPropCache[str];
  }
  return (CssPropCache[str] = str.replace(uppercasePattern, '-$&').toLowerCase() + ':');
}

const ATTRIBUTE_NAME_START_CHAR =
  ':A-Z_a-z\\u00C0-\\u00D6\\u00D8-\\u00F6\\u00F8-\\u02FF\\u0370-\\u037D\\u037F-\\u1FFF\\u200C-\\u200D\\u2070-\\u218F\\u2C00-\\u2FEF\\u3001-\\uD7FF\\uF900-\\uFDCF\\uFDF0-\\uFFFD';

const ATTRIBUTE_NAME_CHAR = ATTRIBUTE_NAME_START_CHAR + '\\-.0-9\\u00B7\\u0300-\\u036F\\u203F-\\u2040';

export const VALID_ATTRIBUTE_NAME_REGEX = new RegExp('^[' + ATTRIBUTE_NAME_START_CHAR + '][' + ATTRIBUTE_NAME_CHAR + ']*$');

const illegalAttributeNameCache = {};
const validatedAttributeNameCache = {};

export function isAttributeNameSafe(attributeName: string): boolean {
  if (validatedAttributeNameCache[attributeName] !== void 0) {
    return true;
  }
  if (illegalAttributeNameCache[attributeName] !== void 0) {
    return false;
  }
  if (VALID_ATTRIBUTE_NAME_REGEX.test(attributeName)) {
    validatedAttributeNameCache[attributeName] = true;
    return true;
  }
  illegalAttributeNameCache[attributeName] = true;
  if (process.env.NODE_ENV !== 'production') {
    // tslint:disable-next-line:no-console
    console.log('Invalid attribute name: ' + attributeName);
  }
  return false;
}

export const voidElements = new Set([
  'area',
  'base',
  'br',
  'col',
  'command',
  'embed',
  'hr',
  'img',
  'input',
  'keygen',
  'link',
  'meta',
  'param',
  'source',
  'track',
  'wbr'
]);

export function createDerivedState(instance, nextProps, state) {
  if (instance.constructor.getDerivedStateFromProps) {
    return combineFrom(state, instance.constructor.getDerivedStateFromProps(nextProps, state));
  }

  return state;
}

export function renderFunctionalComponent(vNode: VNode, context) {
  const props = vNode.props || EMPTY_OBJ;
  return vNode.flags & VNodeFlags.ForwardRef ? vNode.type.render(props, vNode.ref, context) : vNode.type(props, context);
}

redpajama_set_name: RedPajamaGithub


FRANKFURT, April 1 — Financial markets have not "priced in" the risk of a no-deal Brexit and could suffer turmoil if the "worst-case scenario" materialises, top European central bankers warned today.
The risk of Britain crashing out of the EU on April 12 is "something that we cannot neglect," European Central Bank vice-president Luis de Guindos told European Parliament lawmakers in Brussels.
"Markets have not priced in the possibility of a no-deal scenario… just suppose that this worst-case scenario arrives, we have to be prepared," he added.
European institutions and national governments "have taken actions… to minimise the potential impact" while "the private sector is more prepared… than it was only six months ago," de Guindos said.
But with the eurozone economy slowing late last year and into the start of 2019, a disorderly Brexit "could be a sort of amplifier of the negative impacts and the downward trends" by undermining confidence still further, he said.
"Markets sometimes overshoot" in reacting to political events, de Guindos noted.
The former Spanish economy minister's warning followed up a similar statement from Dutch central bank chief Klaas Knot.


Thread: Get Internet Download Manager license FREE!
We are pleased to offer 20 licenses of the latest Internet Download Manager v 5.18, FREE to the readers of The Windows Club !
Version 5.18 adds IDM download panel for web-players that can be used to download flash videos from sites like YouTube, MySpaceTV, and Google Videos and supports Windows 7 and Vista.
Internet Download Manager costs $29.95, but 10 licenses are being made available free to you, the members of The Windows Club Forum. To win one simply make a request below stating why you need one. The winners will be selected randomly, an your mail id's forwared to the company, who will then forward the license to you. TWC readers have been reserved another 10 licenses here.
The giveaway will be kept open for a week.
Special thanks to my MVP colleague Ankur for suggesting that I contact IDM; who were gracious enough to sponsor the 20 licenses for TheWindowsClub.com.
niel28 and Blasius like this.
saurav and karansaini like this.
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I used different kinds of download manager before because I only have a 384kbps internet connection speed. Download manager is a must to manage downloads especially with resume support. And Internet Download Manager is the one who noticeable speed among the rest. I only used freeware for managing my download.
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