text:
In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.
Innate and acquired immunities are both essentially required for the development of optimal resistance to pathogenic fungi (1). The host immune system is a major determinant of which particular form of disease will develop after exposure to the ubiquitous Aspergillus fumigatus or whether transition from saprophytism to infection will occur with the human commensal Candida albicans (2, 3, 4). These fungi are sensed by the innate immune system in a morphotype-specific fashion; that is, different effector mechanisms of immunity are elicited by the different fungal morphotypes (5, 6, 7). Dendritic cells (DCs)3 showed a remarkable functional plasticity in response to the different forms of fungi, being able to discriminate between the different forms in terms of maturation, cytokine production, and induction of Th cell reactivity in vitro and in vivo (5, 6, 8, 9). Different receptors on DCs were found to participate in the fungal recognition event, either independently or through receptor cooperativity (4, 5, 6, 10).
Toll-like receptors (TLRs) are a family of conserved, mammalian cellular receptors that mediate cellular responses to structurally conserved pathogen-associated microbial products (11, 12). All TLRs activate a core set of stereotyped responses, such as inflammation (13). However, individual TLRs can also induce specific programs in cells of the innate immune system that are tailored for the particular pathogen (12, 13, 14). TLRs bear homology to the IL-1R type 1 (IL-1RI) and share a similar signaling cascade culminating in activation of NF-κB and mitogen-activated protein kinases (15, 16). This process facilitates the transcription of genes regulating the inflammatory and adaptive immune responses. The common signal pathways used by IL-1RI and TLRs involve recruitment of the adapter protein myeloid differentiation primary response gene 88 (MyD88) through the homophilic interaction of the Toll/IL-1R homology domain (17). MyD88, in turn, activates a series of IL-1R-associated kinases that are crucially involved in innate immunity. MyD88-deficient mice do not make TNF-α in response to activators of TLR2, TLR3, TLR4, or TLR9, and IL-1R-associated kinase-deficient mice are impaired in their response to LPS and IL-1 (18). However, in the case of TLR4-dependent LPS signaling, another protein (Mal/TIRAP) may also serve as an adapter molecule with or in place of MyD88 (19, 20). It is recognized that the intricacies of how TLRs signal will ultimately provide an explanation for the molecular basis of how cells involved in innate immunity dictate the processes of host defense specific to the provoking pathogen (14).
In the case of fungi, in vitro studies have shown that Cryptococcus neoformans, C. albicans, and A. fumigatus may signal through TLRs, particularly TLR2, TLR4, and TLR9, in a morphotype-specific fashion (21, 22, 23, 24, 25, 26, 27, 28). This will not come as a surprise, given the initial discovery of the Toll pathway in Drosophila as an essential mechanism of antifungal resistance (29). However, because of the complexity of TLR activation and function, including TLR cooperativity (30), the need for coreceptor (24), the sharing of common signal transduction pathways (31), and the dynamic regulation of TLR expression in vivo (32), the in vitro studies may not be sufficient to predict the impact of the total repertoire of TLRs on infections in vivo. In addition, the use of inactivated fungi in the in vitro assays cannot anticipate the impact on TLR functioning of fungal morphogenesis, which is known to be activated in vivo by a wide range of tissue-specific signals (33) and to affect host responses (4, 34).
In the present study we have taken a comprehensive approach to define the role of the IL-1R/TLR/MyD88-dependent signaling pathway in response to C. albicans and A. fumigatus. We evaluate the course and outcome of the infections together with parameters of innate and adaptive antifungal immunity in IL-1RI-, TLR2-, TLR4-, TLR9-, and MyD88-deficient mice with experimental models of infection in which the contributions of innate and adaptive immunities are well established (35). We found that the IL-1R/TLR/MyD88 system plays a strategic role at the interface between the host and fungi, having an impact on the generation of both host antifungal immunity and fungal infectivity.
Materials and Methods
Female, 8- to 10-wk-old C57BL6 mice were obtained from Charles River Breeding Laboratories (Calco, Italy) or The Jackson Laboratory (Bar Harbor, ME). Breeding pairs of homozygous TLR2- (36), TLR4- (37), TLR9- (38), IL-1RI- (39), and MyD88- (40) deficient mice, raised on the C57BL6 background, were bred under specific pathogen-free conditions at the breeding facilities of University of Perugia (Perugia, Italy) or Boston University Medical Center. All in vivo studies were performed in compliance with national, Perugia University, and Boston University animal care and use committee guidelines.
Microorganisms, culture conditions, and infections
Isogenic strains of C. albicans, obtained by mutagenesis in vitro and capable (Vir−13), or not (Vir−3), of yeast-to-hyphal transition, as assessed by germ-tube formation in vitro, were used (41). The A. fumigatus strain was obtained from a fatal case of pulmonary aspergillosis at the Infectious Diseases Institute of University of Perugia (41). Yeasts were harvested at the end of the exponential phase of growth and resuspended in IMDM (Life Technologies Italia, Milan, Italy) containing 5 μg/ml polymyxin B (Sigma-Aldrich, St. Louis, MO) and 50 μg/ml gentamicin. Conidia were harvested by extensive washing of the slant culture (on Sabouraud dextrose agar (Difco, Detroit, MI) supplemented with chloramphenicol for 4 days at room temperature) with 5 ml of 0.025% Tween 20 in saline. For generation of hyphae, Candida cells were allowed to germinate by culture at 37°C in 5% CO2 for 2 h in RPMI 1640 medium (by that time, >98% of cells had germinated). For Aspergillus infection, conidia were given intranasally (i.n.) for 3 consecutive days (2 × 107/20 μl saline/each injection) as previously described (6). Mice were anesthetized by i.p. injection of 2.5% avertin (Sigma-Aldrich). For primary disseminated candidiasis, mice were injected i.v. with 106/0.5 ml of C. albicans yeasts or hyphae; for reinfection, mice surviving the primary infection were injected i.v. with 106/0.5 ml of C. albicans hyphae 14 days later (41). For the gastrointestinal infection, 108 Candida hyphae were injected intragastrically (i.g.), via an 18-gauge, 4-cm-long plastic catheter in a volume of 0.2 ml saline/mouse as previously described (35). Cyclophosphamide (Sigma-Aldrich) was given at 150 mg/kg i.p. 1 day before challenge. Quantification of fungal growth in the organs of infected mice was performed by plating serial dilutions of homogenized organs in Sabouraud dextrose agar, and results (mean ± SE) were expressed as CFU per organ.
Cell purification
Polymorphonuclear neutrophils (PMNs) were isolated from the blood and peritoneal cavity of mice as previously described (35). Briefly, peritoneal PMNs were obtained 8 h after the i.p. injection of 1 ml of endotoxin-free 10% thioglycolate solution (Difco). Endotoxin was depleted from all solutions with Detoxi-gel (Pierce, Rockford, IL). To purify Gr-1+ PMN, 107 cells were incubated with biotin-conjugated anti-mouse Gr-1 mAbs (clone RB6-8C5; BD PharMingen, San Diego, CA) for 30 min at 4°C and then with avidin-conjugated magnetic MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) for 15 min at 6°C, and magnetically separated with a positive selection column (Miltenyi Biotech) according to the manufacturer's instructions. On FACS analysis, Gr-1+ PMN were >98% pure and stained positively for the CD11b myeloid marker. Cytospin analysis confirmed that the population consisted of polymorphonuclear cells (>98%). Murine CD11c+ DCs were isolated from spleens and lungs, and CD4+ T cells were isolated from mesenteric lymph nodes (MLN), thoracic lymph nodes (TLN), and spleens by magnetic cell sorting as previously described (5, 6, 8, 9).
Phagocytosis, cultures, and antifungal effector activity
For phagocytosis, peritoneal neutrophils from infected mice were incubated at 37°C with unopsonized Candida yeasts or Aspergillus conidia for 30 and 60 min, respectively, at an effector to fungal cells ratio of 1:3. After incubation, phagocytic cells were separated from nonphagocytosed fungal cells by centrifugation on an FBS gradient, and a 0.1-ml sample of the harvested phagocytic cells was used for cytospin preparation. The percentage of internalization was calculated on Giemsa-stained preparations as previously described (5, 6). For staining of degranulated PMNs, the cytospin preparations were subjected to methylene blue (for azurophil granules) or eosin (for nonazurophil granules) staining following standard procedures. For myeloperoxidase detection, the liquid diaminobenzidene substrate Pack Kit (BioGenex, Menarini Diagnostics, Florence, Italy) was used according to the manufacturer's instructions on ematoxilin-counterstained preparations. For the fungicidal activity against Candida yeasts or Aspergillus conidia, PMNs were incubated with unopsonized fungal cells (at an effector to fungal cell ratio of 1:5) for 60 min (for yeasts) or 120 min (for conidia) at 37°C. Triton X was then added to the wells, and serial dilutions from each well were made in distilled water. Pour plates (four to six replicate samples) were made by spreading each sample on Sabouraud glucose agar. The number of CFUs and the percentage of CFU inhibition (mean ± SE), referred to as candidacidal or conidiocidal activity, were determined as previously described (5, 25). For the fungicidal activity against hyphae, a colorimetric MTT assay was used as previously described (42, 43). Briefly, graded numbers of PMNs (106, 5 × 105, and 105 5 yeasts in 96-well, flat-bottom microtiter plates. After 2 h at 37°C with occasional shaking (by that time, >98% of cells had germinated), the supernatants were aspirated, effector cells were lysed by adding sodium deoxycholate (0.5%), and hyphal viability was determined by MTT staining. Antifungal activity was calculated according to the following formula: percentage of hyphal damage [(1 − x)/C] × 100, where x represents the OD of test wells, and C represents the OD of control wells with hyphae only. Each set of conditions was tested in duplicate, and the results were averaged. PMN production of reactive oxygen intermediates (ROI) was performed by quantifying the superoxide anion (O2−) production by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c as previously described (44). Peripheral PMNs (2 × 106) were incubated in the presence of 80 μM cytochrome c with 2 × 107 unopsonized yeasts or conidia for 60 or 120 min, respectively, at 37°C. A550 was measured in the culture supernatants in a Microplate Reader 550 (Bio-Rad, Rome, Italy), and the background absorbance in control tubes containing only buffer and cytochrome c was subtracted. Experiments were performed in triplicate, and the results were expressed as nanomoles of O2−. The effects of TLR stimulation on fungicidal activity, respiratory burst, and degranulation were assessed by preincubating PMNs for 120 min with the different TLR ligands (zymosan (10 μg/ml) and lipoteichoic acid (LTA; 1 μg/ml) from Staphylococcus aureus and LPS (10 μg/ml) from Salmonella minnesota Re 595 (all from Sigma-Aldrich) and unmethylated CpG oligonucleotide 1826 (2 μM) of proven immunostimulatory sequence (25)) before the addition of unopsonized yeasts or resting conidia. Photographs were taken using a high resolution microscopy color camera (AxioCam Color), using the AxioVision software Rel. 3.0 (Carl Zeiss, Milan, Italy). For cytokine determination, purified DCs were resuspended in IMDM with no serum, but with polymixin B, to avoid nonspecific activation by serum components and endotoxin and were pulsed with unopsonized Candida yeasts and hyphae or Aspergillus resting conidia for 2 h before the addition of amphotericin B to prevent fungal overgrowth as previously described (5, 6). Cytokine measurement was performed after an additional 22 h of coculture. The levels of cytokine production by DCs added with amphotericin B alone were below the detection limits of the assays (data not shown).
Proliferation assay by flow cytometric analysis
Proliferation of 1 × 106 splenic CD4+ T lymphocytes stimulated with 5 × 105 Ag-pulsed splenic DCs for 5 days at 37°C was assessed by labeling with CFSE (Molecular Probes, Eugene, OR) as previously described (9).
Cytokine and ELISPOT assays
5, 6, 9). Results are expressed as the mean number of cytokine-producing cells (±SE) per 105 cells, calculated using replicates of serial 2-fold dilutions of cells.+ T cells, an ELISPOT assay was used on purified CD4+ T cells as previously described (
Statistical analysis
The log-rank test was used for paired data analysis of the Kaplan-Meier survival curves. Student's t test or ANOVA and Bonferroni's test were used to determine the statistical significance of differences in organ clearance and in vitro assays, as indicated in the figure legends Significance was defined as p < 0.05. In vivo groups consisted of four to six animals. The data reported were pooled from three to five experiments unless otherwise specified.
Impact of the IL-1R/TLR/MyD88-dependent signaling pathway to susceptibility to candidiasis and aspergillosis
To assess the role of the IL-1R/TLR/MyD88-dependent pathway in the course and outcome of candidiasis and aspergillosis, TLR2-, TLR4-, TLR9-, IL-1RI-, and MyD88-deficient mice were infected with Candida yeasts or hyphae i.v., Candida hyphae i.g., or Aspergillus conidia i.n. Mice were then assessed for survival and fungal growth in the kidneys, stomach, and lungs, respectively. Similar to wild-type mice, TLR2-, TLR4-, and TLR9-deficient mice survived the infection with low virulence Candida yeasts, in contrast to IL-1RI- and MyD88-deficient mice, which succumbed to it (Fig. 1⇓A). Susceptibility to virulent Candida hyphae given i.v. was not different among the different types of mice, in that all succumbed to the infection, although TLR4- and TLR9-deficient mice survived significantly longer (Fig. 1⇓B). For aspergillosis, although each type of intact mouse survived the infection (data not shown), upon immunosuppression with cyclophosphamide there were important differences among strains, as TLR-4- and MyD88-deficient mice died sooner, and IL-1RI-deficient mice died later than the other strains (Fig. 1⇓C). We also assessed the susceptibility to reinfection with Candida hyphae of mice surviving the primary infection with Candida yeasts and found that TLR-9-deficient and wild-type mice survived reinfection, whereas the majority of the TLR2- and TLR4-deficient mice succumbed (Fig. 1⇓D). Resistance to reinfection of intact mice surviving the Aspergillus primary infection was also reduced in TLR2-deficient mice (data not shown).
The assessment of the actual fungal growth in the relevant target organs correlated with the pattern of survival. The fungal burden was significantly higher in IL-1R1- and MyD88-deficient mice highly susceptible to Candida yeasts compared with the other types of mice, although it was significantly lower in resistant TLR-9- and TLR-2-deficient mice (Fig. 2⇓A). Fungal growth in the kidneys of mice infected with Candida hyphae i.v. was not different among the different types of mice, except for the significant reduction in TLR9- and TLR2-deficient mice (Fig. 2⇓B); however, it was different in the stomach after i.g. infection, as shown in Fig. 2⇓C. The fungal burden was significantly lower in resistant TLR9-deficient mice and was significantly higher in TLR2- and TLR4-deficient mice, particularly in IL-1RI- and MyD88-deficient mice. No signs of dissemination to visceral organs, such as esophagus, small intestine, liver, and kidneys, were observed. Moreover, the fungal burden continued to be elevated 2 wk after the infection, but started to decline at 4 wk, and all mice eventually survived the infection (data not shown). For Aspergillus, the growth of the fungus was significantly lower in the lungs of TLR9- and IL-1RI-deficient mice, but was higher in TLR2-, TLR4-, and MyD88-deficient mice compared with wild-type mice (Fig. 2⇓D). Histological analysis of infected organs revealed the presence of numerous fungal elements in the relative absence of signs of inflammatory pathology in IL-1RI-, TLR4-, and MyD88-deficient mice (data not shown). Together, these results show that the expression of host resistance to fungi may occur through common as well as divergent patterns of TLR activation depending on fungal species and morphotype as well as the route of the infection. IL-1RI and TLR4 were required for resistance to the primary infection with Candida and Aspergillus, respectively, and the MyD88-dependent pathway was required for resistance to both. TLR2 and TLR4 were required for resistance to reinfection; the requirement of TLR4 for resistance to Candida was dependent on the type of infection (mucosal vs disseminated candidiasis). Finally, TLR9 was not required for resistance to either fungus. In addition, the ability of the mutant mice to survive the Aspergillus infection if intact suggests that resistance to the fungus may also occur through compensatory TLR-independent mechanisms.
Impact of the IL-1R/TLR/MyD88-dependent signaling pathway on the innate antifungal response
To correlate the above findings with patterns of innate antifungal resistance, we measured the levels of TNF-α production early in the course of infection in kidneys and lungs of mice with candidiasis and aspergillosis, respectively, because this cytokine is required for resistance to both fungi (35) and is prototypically released through TLR-dependent pathways (12, 13, 17). We also assessed the antifungal effector activities of PMNs, as they are essential components of the innate resistance to fungi (45, 46). The levels of TNF-α were particularly elevated in TLR2-deficient mice, similar to control mice in TLR9-deficient mice, and were ablated in MyD88-deficient mice infected with either fungus (Fig. 3⇓). However, fungal species-specific differences did emerge, as TNF-α levels were lower in TLR4- and IL-1RI-deficient mice infected with Candida, but not Aspergillus. Therefore, TNF-α production was not predictive of resistance or susceptibility to infections, as neither was associated with a specific TLR repertoire. However, TNF-α production was crucially dependent on the MyD88 pathway.
To evaluate PMNs, we assessed their ability to phagocytose and kill fungi, the expression of TLR upon exposure to fungi, and the functional responses to known TLR ligands. As the recruitment of PMNs at the sites of the infection, i.e., the kidneys, stomach, and lungs, was slightly decreased in TLR2- and TLR4-deficient mice only (as judged by cytospin analysis; data not shown), we assessed PMNs from infected and uninfected mice for functional antifungal activity, such as the ability to internalize and kill both fungi. Similar to what we observed with PMNs from uninfected mice, differences were found in the ability to handle fungi by PMNs from the different types of mice upon infection. The fungicidal activity determined in vitro mirrored the in vivo susceptibility to the primary infections (Fig. 4⇓A). Phagocytosis was slightly impaired, and killing activity was virtually abolished in PMNs from MyD88- and IL-1R1-deficient mice against Candida yeasts; in the case of Aspergillus conidia, phagocytosis and killing were impaired in MyD88-deficient and particularly in TLR4-deficient PMNs. In contrast, TLR2 and TLR9 deficiencies did not affect the ability to internalize and kill either fungus, except for the slight impairment of the conidiocidal activity observed in TLR2-deficient PMNs. In the case of Candida hyphae, the antifungal effector activity of PMNs from uninfected mice was significantly increased in TLR4- and TLR9-deficient mice and was impaired in MyD88- and IL-1RI-deficient mice. Because cytokine production during the infection may have an impact on the antifungal functions of PMNs, and TNF-α is known to selectively impair the activity of PMNs against Candida hyphae (43), the effector function of PMNs was also assessed in the presence of TNF-α. The ability of hyphal damage was greatly reduced by TNF-α in TLR4-deficient PMNs and was slightly reduced in PMNs from TLR9-deficient or wild-type mice. No effect was observed on PMNs from the other types of mice, including MyD88-deficient mice which, similar to TLR4-deficient mice, showed defective TNF-α production during the infection (Fig. 4⇓A). Therefore, additional mechanisms, besides TNF-α production, might regulate the expression of the antifungal effector functions of PMNs at the site of infection.
To correlate these findings with the possible functional activity of TLRs on PMNs, purified PMNs from uninfected wild-type mice were exposed to Candida yeasts and hyphae or Aspergillus conidia and hyphae and were assessed for TLR2, TLR4, and TLR9 expression by RT-PCR. In addition, as the ability of PMNs to kill fungi occurs through both oxidative and nonoxidative mechanisms (47), we also assessed the antifungal effector activity of PMNs upon exposure to the relevant TLR ligands in terms of ROI production and degranulation. We found that the expression of both TLR2 and TLR9 was induced upon exposure to viable unopsonized yeasts or conidia, but not to hyphae; however, TLR4 expression was induced upon exposure to each fungal morphotype (data not shown). In terms of the functional consequences of stimulation of the relevant TLRs on PMNs, CpG did not significantly modify the antifungal effector activity against both yeasts and conidia; LTA completely inhibited the fungicidal activity of PMNs, whereas LPS and zymosan increased it (Fig. 4⇑B). Activation of the respiratory burst leading to the generation of ROI was observed in response to yeasts and, to a lesser extent, in response to resting conidia. Similarly, degranulation, as observed upon eosin staining of nonazurophil granules (pink staining) or methylene blue staining of azurophil granules (blue staining), was minimal upon stimulation with resting conidia compared with that of yeasts. Stimulation with zymosan and LTA maximally stimulated the production of ROI against both fungi, but LPS and CpG did not (Fig. 4⇑C). In contrast, LPS induced the extracellular secretion of azurophil granules in response to both fungi to an extent superior to that in wild-type mice, either unstimulated or stimulated with zymosan, LTA, or CpG. Interestingly, PMN stimulated with LPS also stained positively with myeloperoxidase (Fig. 4⇑C, inset). Together, these data indicate that the expression of the innate immune response to fungi occurs through the involvement of distinct members of the IL-1R1/TLR superfamily, each probably activating specialized antifungal effector functions on PMNs and implicating the MyD88-dependent signaling pathway. Furthermore, they confirm that oxidant generation by PMNs may not always be sufficient to mediate fungal killing without complimentary nonoxidative mechanisms, as previously suggested (46, 47, 48).
Impact of the IL-1R/TLR/MyD88-dependent signaling pathway on antifungal adaptive immunity
To define the impact of the IL-1R/TLR/MyD88-dependent signaling pathway on the quality of the adaptive immunity to fungi, we determined the frequencies of IFN-γ- or IL-4-producing CD4+T lymphocytes in uninfected mice or mice with gastrointestinal candidiasis or pulmonary aspergillosis. To this purpose, mice were infected i.g. with Candida hyphae or i.n. with Aspergillus conidia, and the number of Th1 or Th2 cytokine-producing cells was assessed in the MLN and TLN, respectively, 1 wk after the infection. No differences were observed in the frequencies of cytokine-producing cells among the different types of uninfected mice. However, the number of cells producing IFN-γ was significantly reduced and that of cells producing IL-4 was significantly increased in MyD88-deficient mice with either infection. A similar pattern of Th1/Th2 reactivity was observed in TLR9-deficient mice despite their remarkable antifungal resistance. Th2 cells predominated over Th1 cells in IL-1R-deficient mice with candidiasis, but not with aspergillosis. Finally, evidence of activation of both Th1 and Th2 cells was found in TLR2- or TLR4-deficient mice with candidiasis and aspergillosis, as the number of IL-4-producing cells was increased, and the number of cells producing IFN-γ was either decreased, in candidiasis, or remained unchanged, in aspergillosis, compared with that in control mice (Fig. 5⇓).
As DCs are instrumental in shaping the adaptive Th response to fungi (4, 9, 49), we assessed IL-12 p70/IL-10 production by purified DCs from each type of mouse in response to yeasts, conidia and hyphae. The ability to phagocytose either fungus was not significantly different between DCs from mutant mice and those from wild-type mice (data not shown); however, their abilities to produce cytokines were different. The production of IL-12 p70 was virtually ablated in DCs from MyD88-deficient mice against each fungal morphotype, and this was associated with a significantly increased production of IL-10. A similar cytokine pattern was observed with IL-1RI-deficient DCs in response to Candida yeasts and in TLR-4-deficient DCs in response to Aspergillus conidia. An opposing pattern was observed in TLR2- and TLR9-deficient mice. The production of IL-12 p70 was increased and that of IL-10 was decreased in TLR2-deficient DCs in response to yeasts and conidia, and the reverse was true with TLR9-deficient DCs. It is of interest that although the production of IL-12 p70 in response to hyphae was only reduced in MyD88-deficient DCs, the production of IL-10 was significantly increased in MyD88-deficient mice, but, interestingly, was significantly decreased in TLR2-, TLR4-, and TLR9-deficient DCs (Fig. 6⇓A). Together these results suggest that the production of cytokines by DCs in response to fungal morphotypes is dependent on the type of receptor involved, even though signaling through MyD88 is essentially required for IL-12 p70, but, interestingly, not for IL-10 production. Criss-cross experiments in which the proliferative response of CD4+ T cells from MyD88-deficient or wild-type uninfected mice was assessed in the presence of fungus-pulsed DCs from MyD88-deficient or wild-type mice confirmed the crucial role of MyD88-dependent signaling on DCs for antifungal Th1 priming. Lymphoproliferation was observed in cultures of lymphocytes with DCs from wild-type mice, but not from MyD88-deficient mice (Fig. 6⇓B). However, the proliferative capacity of lymphocytes from wild-type mice was lost, and that of lymphocytes from MyD88-deficient mice was gained in the presence of DCs from mutant or wild-type mice, respectively (Fig. 6⇓B). High levels of IFN-γ production and no IL-4 were detected in culture supernatants of proliferating cells (data not shown).
This study describes the impact of the IL-1R/TLR/MyD88 system on the host response to C. albicans and A. fumigatus in vivo. Although in vitro studies have indicated the importance of TLR signaling in response to Candida and Aspergillus (21, 22, 23, 24, 25, 26, 27, 28), the role of TLRs in the generation of innate and adaptive immunity to fungi remains largely undefined. We show in this study that 1) the MyD88-dependent pathway is essentially required for the innate and Th1-mediated resistance to either fungus; 2) the involvement of MyD88 may occur through signaling by distinct TLRs depending upon the fungal species, morphotypes, and site of infection; and 3) TLRs may contribute differently to the occurrence of innate and adaptive Th1 immunities to each fungus, consistent with the ability of each individual TLR to activate specialized antifungal effector functions on PMNs and DCs. However, fungi may also exploit TLRs for fungal growth and survival in vivo, as recently suggested (27).
In line with the idea of the essential role played by MyD88-dependent signaling in the development of Th1 responses (17), the susceptibility of MyD88-deficient mice to candidiasis and aspergillosis was associated with the occurrence of a defective antifungal Th1 response, probably due to defective DC activation. However, as the antifungal effector functions of PMNs were also impaired, MyD88-dependent signaling has a crucial role in the expression of optimal antifungal innate resistance. For Aspergillus, the fact that intact MyD88-deficient mice survived the infection suggests that resistance to the fungus may also occur through MyD88-independent mechanisms. Intriguingly, Drosophila MyD88, although only needed for antifungal defense, is unable to induce expression of the antifungal peptide drosomycin in the absence of other adapters (50). Preliminary data showing the ability of LPS to activate antifungal effector functions in MyD88-deficient mice support this hypothesis. However, because TLR4 is known to signal through both the MyD88-dependent and -independent pathways (14), but TLR4-deficient mice are not more susceptible to aspergillosis than MyD88-deficient mice, further studies are needed to elucidate the role of MyD88-independent pathways in resistance to Aspergillus. It is of interest that the conidiocidal activity, more than the phagocytic ability, was almost completely ablated in TLR4-deficient PMNs, a finding suggesting that internalization of the fungus, in the absence of TLR4 signaling, may contribute to virulence by preventing fungal killing and thus favoring fungal survival.
In candidiasis, in line with the increased susceptibility to the infection seen in TLR4-defective C3H/HeJ mice (23), our study shows that TLR4-deficient mice mount a defective Th1-protective immunity to fungus in the face of an efficient innate antifungal resistance, particularly against hyphae. However, a more crucial role is played by IL-1R1, as both the innate and acquired Th1 resistances to Candida infections were severely impaired in its absence. The finding that, similar to MyD88-deficient mice, IL-1R-deficient mice were highly susceptible to low virulence Candida yeasts qualifies the IL-1RI/MyD88-dependent pathway as a major host determinant of fungal virulence in vivo. The killing ability of PMNs against Candida was also severely impaired in IL-1R-deficient mice, a finding consistent with the importance of IL-1-dependent (51), but more importantly IL-18-dependent (52), signaling in the activation of PMNs to an antifungal state. However, although anticandidal resistance was decreased in conditions of IL-1 (53) and/or IL-18 (54) deficiencies, neither deficiency was as severe as that of IL-1R1-deficient mice in terms of susceptibility to the infection.
TLR2 signaling by zymosan (55), β-glucan (56), Candida phospholipomannan (26), Aspergillus conidia (24, 27), and hyphae (27, 28) led to the production of both inflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines depending on the cell type. Unopsonized zymosan also stimulated the respiratory burst of phagocytes, although opsonization greatly increased this capacity (57). However, the susceptibility of TLR2-deficient mice to primary candidiasis and aspergillosis was not different from that of control mice, a finding suggesting that the mice are fully competent at the level of innate antifungal resistance, as documented by reduced fungal growth in mice with primary disseminated candidiasis. In contrast, they failed to mount protective Th1 resistance, as evidenced by the high susceptibility to mucosal infection and reinfection, both known to be controlled by Th1 adaptive immunity (35). Both Th1 and Th2 lymphocytes were indeed activated in these mice despite the high level production of IL-12 p70 by DCs. The interesting finding was that IL-10 production was greatly reduced in DCs from these mice in response to fungi. Because IL-10-producing DCs are required for the expression of long-lasting memory antifungal immunity through the induction of regulatory T cells (58), it is possible that the Th1 hyporesponsiveness of TLR2-deficient mice is a consequence of the decreased IL-10 production by DCs. Whatever the mechanism is, our finding is consistent with the defective response to vaccination with Borrelia burgdorferi OspA seen in TLR2-deficient mice (36) as well as with the TLR2 dependency of IL-10 production in response to Aspergillus hyphae (27).
The findings with TLR9-deficient mice are interesting, although largely unexplained. As TLR9 stimulation by CpG enhances innate effector (59) and Th1 responses in experimental models of infection (60), including aspergillosis (25), one would reasonably expect decreased resistance to fungi. We found that TLR9-deficient mice were incapable of mounting an Ag-specific Th1 response, yet they were highly resistant to both mucosal candidiasis and reinfection. TLR9-deficient mice were particularly efficient in restricting fungal growth upon primary infection, particularly with Candida hyphae, against which they showed potent antifungal effector activity.
Consistent with their role in immune surveillance, TLRs are expressed at higher levels in tissues exposed to the external environment, such as lung and gastrointestinal tract (32). TLRs have also been shown to be expressed on human peripheral blood PMNs (61) and to mediate antimicrobial responses (59, 62). PMNs can exert antifungal activity by both oxidative and nonoxidative mechanisms (63). The former is characterized by the respiratory burst resulting in the generation of oxidants with potent antimicrobial activity. A multitude of mediators with nonoxidative antifungal activity has been also found in human PMN granules, with the primary (azurophil) granules containing, among others, myeloperoxidase and defensins (64). The quantity and specificity of delivery of these toxic neutrophil products ultimately will determine the relative efficiency of fungicidal activity vs inflammatory cytotoxicity to host cells.
It is well known that the various fungal forms not only elicit differing responses from PMNs and phagocytes, but also differ in their susceptibilities to cellular microbicidal mechanisms (47). We found in this study that murine PMN activation by the different fungal forms occurs through TLR signaling that affected the release of both granule constituents and oxidants more than phagocytosis. This is consistent with previous findings demonstrating roles for zymosan (57), LPS (65), CpG (59), and p38 mitogen-activated kinases (66) in regulating a variety of neutrophil functions, including the production of ROI and degranulation. Consistent with the expression of TLR2, TLR4, and TLR9 on PMNs upon exposure to fungi, zymosan, LPS, and, in part, CpG stimulated the fungicidal activity of PMNs. However, different mechanisms of antifungal activity were elicited. The production of ROI was higher in response to zymosan than LPS or CpG, but degranulation was maximally induced by LPS and less so with zymosan or CpG. Interestingly, azurophilic granules with myeloperoxidase activity were mobilized by LPS more than by zymosan or CpG. Experiments with Candida and Aspergillus hyphae have suggested that degranulation, more than ROI production, correlated with the maximum antifungal effector function of PMNs, an activity that occurs differently in the different types of mice and in response to the different TLR ligands (S. Bellocchio, C. Montegnoli, S. Bozzo, G. Rossi, and L. Romoni, manuscript in preparation). Together these findings, although highlighting the complexity and multitude of mechanisms underlying the different susceptibilities of TLR-deficient mice to the primary infections with each fungal morphotype, also suggest that different TLRs activate specialized antifungal effector functions on PMNs and support the idea that different PMN functions are mediated by divergent transduction pathways (63). Interestingly, stimulation with LTA abrogated the fungicidal activity of PMNs, and this was associated with stimulation of ROI production, but not with degranulation. This finding, although confirming that ROI stimulation without the concomitant activation of granule proteases is not sufficient to mediate antifungal activity (47, 48), also unmasks the effect of TLR cooperativity at the level of PMN activation.
In line with the idea that TLR2 ligands are recognized by heterodimers formed between TLR2 and other TLRs (30), host recognition of different configurations of lipoprotein/lipopeptides occurs through the heterodimeric association of TLR2 with either TLR1 or TLR6 (67). In this regard it is of interest that the capacity of PMNs to kill C. albicans was greatly reduced after exposure to medium, but not long, chain fatty acid-containing lipid emulsions in vitro (68) and in vivo (69). As similar results, in terms of TLR expression and functioning, have been obtained with human PMNs (S. Bellocchio, C. Montegnoli, S. Bozzo, G. Rossi, and L. Romoni, manuscript in preparation), together these data indicate that the expression of the innate response to fungi occurs through the involvement of distinct TLRs, each probably activating specialized antifungal effector functions on PMNs and implicating the MyD88-dependent signaling pathway. Importantly, the finding that TLRs affect the balance between fungicidal oxidative and nonoxidative mechanisms of PMNs suggests that TLR manipulation in vivo may be amenable to the induction of optimal microbicidal activity in the absence of inflammatory cytotoxicity to host cells.
It is still unclear whether pathogen-specific Th2 responses develop by default in the absence of the TLR/MyD88-dependent signal or whether a specialized class of recognition receptors activates Th2 responses upon recognition of Th2-specific pathogens (12, 14). This issue may be particularly relevant for the understanding of how Th2 immune responses are generated to fungal hyphae. We have recently shown that additional receptors of fungi, such as Fc, complement and lectin receptors, contributed to the generation of Th1/Th2 responses to the different fungal forms (4). The relation between TLRs and these receptors is largely unknown at the moment. The data from the present study show that the TLR repertoire contributes to the functional plasticity of DCs in response to the different fungal morphotypes, including hyphae. MyD88-deficient DCs were clearly impaired in their ability to activate Th1 cells. However, as no obvious correlation could be found between patterns of cytokine production by DCs and type of antifungal Th reactivity, such as in conditions of TLR2 or TLR4 deficiency, these findings indicate the existence of additional dysfunctional activities at the DC level, as previously suggested (70).
In conclusion, this study shows the diversity and complexity of the signaling pathways triggered by TLRs in response to fungi in vivo (summarized in Table I⇓). Members of the TLR/IL-1R superfamily contributed to the generation of host immunity to C. albicans and A. fumigatus in a manner that is dependent on fungal species and morphotypes as well as the site of infection. Basically, the IL-1R1/MyD88-dependent pathway is essentially required for host resistance to Candida, whereas the TLR4/MyD88-dependent pathway is crucially involved in resistance to Aspergillus. However, as fungal morphogenesis in vivo is activated by a variety of tissue-dependent stimuli (33), this idea may accommodate the finding that TLRs are differently implicated depending on the site of the infection, as clearly shown in infections with Candida hyphae, and suggests that recruitment of multiple TLRs may occur during infection. Finally, the increased antifungal resistance observed under conditions of TLR deficiency not associated with impaired immune resistance suggests that TLRs may be exploited by fungi for survival in vivo.
We thank Dr. Lara Bellocchio for superb editorial assistance, and Dr. Fabrizio Stracci (Department of Hygiene and Public Health, University of Perugia, Perugia, Italy) for assistance with the statistical evaluation. We thank Dr. S. Akira (Department of Host Defense, Osaka, Japan) for the generous supply of breeding pairs of TLR2-, TLR4-, TLR9-, and MyD88-deficient mice obtained through the courtesy of Drs. E. Bates and G. Trinchieri (Laboratory for Immunological Research, Schering-Plough, Dardilly, France). We thank Giorgio Trinchieri for critically reviewing this manuscript and for helpful suggestions.
↵1 This work was supported by the National Research Project on AIDS, Contract 50D.27 (Opportunistic Infections and Tuberculosis, Italy); Project 1AF/F (Immunotherapeutic Approaches in Fungal Infections) from Istituto Superiore di Sanitá (Italy); and National Institutes of Health Grants AI37532 and AI25780.
↵2 Address correspondence and reprint requests to Dr. Luigina Romani, Department of Experimental Medicine and Biochemical Sciences, Microbiology Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail address: lromani{at}unipg.it
↵3 Abbreviations used in this paper: DC, dendritic cell; i.g., intragastrically; IL-1RI, IL-1R type 1; i.n., intranasally; LTA, lipotheicoic acid; MLN, mesenteric lymph node; MyD88, myeloid differentiation primary response gene 88; PMN, polymorphonuclear neutrophil; ROI, reactive oxygen intermediate; SOD, superoxide dismutase; TLN, thoracic lymph node; TLR, Toll-like receptor; Zym, zymosan.
- Received October 6, 2003.
- Accepted December 23, 2003.
- Copyright © 2004 by The American Association of Immunologists


text:
We Are Point2 'APPROVED'
And we are SSL Certificate Friendly ... It is very important as well for REALTORS to have a 'Secure' website when it comes to viewer trust and sharing information on your website. Point 2 recognizes the importance of this and is in the conversion process NOW!
Is YOUR website provider and website SSL Secure?
Start your Point2 Website FREE TRIAL HERE
Break the Mold. Attract Attention
"Finding the person who will be able to interpret what we need especially at a distance is not easy, the many options available do not help either. I cannot be more grateful for having found Cher, the work she has done has been fantastic and I can already see the increase in traffic and quality of prospects to my Point2 website. Thank you very much Cher for your wonderful work and warm attention, with such great service and you did an amaizing job!"
Thank you so much
A. Kleinow - Costa Rica
"After working with 3 web designers, we were thrilled to find Get A Grip. We needed a total redesign. I called the company and talked with both Barry and Cher at length. I immediately felt comfortable with them. Through the entire process, Cher was available by phone and email and responded to all calls and emails in a timely manner. We highly recommend Get a Grip for their creativity, knowledge and customer service. You will not be disappointed."
Kim Odell - Florida
Are you ready? If so "EMAIL US"and let us help you take the first step to a more successful
'Return On Your BUSINESS'
Janice Ponce Royal Lepage
"I am very pleased to say I finally found what I was looking for in website design. A big thank you to Cher & Barry at 'Get A Grip Audio & Website Solutions' (website designs). Mine was long overdue and Barry gently kept on me (for a year!) to talk to Cher about what she could do for me. She listened to every word, all my wants and needs and ran with it. From the colors to pictures to fonts and more, I love it! I have had people call me from all over to tell me how much they love my new website & it's only been up two days! A wealthy friend of mine always says "It takes money to make money," it's very true, but I also believe it takes a team of great people to support you.
Thank you so much 'Get A Grip Audio & Website Solutions' fantastic job!
Janice Ponce
"It's more than I had ever thought it could be. It is amazing how you took the time to listen, to get to know me, to develop an idea of my personality and then deliver it so perfectly through your design of the website!!! I am amazed at your talent and your creativity! Thank you, thank you, thank you!!!"
The Best Regards,
Cynthia Lloyd
Re/Max Pembroke Realty Ltd.
Point2 Website Design by Get A Grip Audio & Website Solutions

subset: commoncrawl


text:
This is the product page for: PETITE Tan Zebra Print High Neck Top
Image carousel, press Enter to cycle through the images, press the Z key to zoom in and out of an image. Once zoomed in, use WASD keys to pan around image.
PETITE Tan Zebra Print High Neck Top
Nail this seasons animal trend in our zebra print high neck top.
Model wears: UK 8
Your shopping bag is empty.
You are viewing the website for the UK. Would you like to view the website for United States instead?
Content is loading, please wait.


text:
Love too much?
Here I am again. You can see this is all troubling me, as I keep on writing here. That is me though. When my mind is racing through so many things at once and I can't make anything of it, I sit down and write (in my journal or here at my blog site). And sometimes it helps me immediately and other times… I keep on having too many thoughts and need to write more.
After my last post I have been wondering if it is possible to love too much? Can you be blinded by love? Can you simply care too much for someone that you will let them use you again and again (not saying that's the case here though, just wondering)? I have read and heard about relationships that I would quickly run away from if it was me in it. But I also can see that when you are crazy about someone, depended maybe, scared that you won't find anyone (better) if you walk away from this one… It is hard to just let go…Most people want to fight. They don't want to let go something that they put so much time and effort in. People want to show to others and/or themselves that they didn't just let it go. That they did the best they could and that it just wasn't enough. But because they fought hard for it, in the end, they can say they tried. And that can be harder than "just" walking away. But when you love someone so much, when you need someone so terribly much, it seems harder to make the "right decision" and "simply" walk away. It both takes guts in my opinion.
But yes, I am wondering if one can love too much. Because that would make one vulnerable and easy to be taken advantage of by the one(s) that one loves/cares about. And of course, there are times when my mind wonders if I might be one of the ones I mentioned a few lines ago. Am I wanting to give too much? Am I too depended from the ones I love?
Every time I see (and if you read my last post then you know I often see just a little too much) something that I don't trust, I start worrying. It could be a simple message from someone BF/GF knows on those popular community sites to a stupid text message on their cellphone they receive. I am not one who sends hearts and kisses to everyone I message. I only send hearts to my BF and GF because well, they are my loves. Only to real, dear friends will I send hugs and kisses. But no hearts and all… OK, I must admit, I have used hearts to only some dear and special friends when they were in need of some love. But only then… (the friends who have received the hearts will know why and all so…).
So when my eye catches comments/tweets/posts from others to my loved ones or the other way around, I immediately see those others as treats. They are getting the loving hearts I should be receiving..! Why are they sending/receiving hearts from "friends" who, as far as I know, are definitely NOT their lovers (seeing I am one of the two lovers in their lives, I was kinda hoping I knew them all…by heart 😉 ). I already start going back in time, "checking" if there were more signs leading up to the point where the hearts were being exchanged… I wonder why they send the hearts, I wonder why someone else would feel the need to send them hearts. Also, when you're just friends, I never send many kisses to them when I send a message. I normally end it with "Liefs" (that's Dutch, of course… It's something like "With love" but in a friendship kinda way, not a love-love kinda way) and then my name. Or just "Groetjes" ("Greets" in Dutch) when they are "just" friends/acquaintances. No amounts of kisses and or hugs… Maybe a hug or two when they are having a hard time but well… I guess I have been used too much during my childhood so that now, as an adult, I am more careful.
I know I can be silly in that way though. I'd rather go to a convention where I know maybe 0,001% of the attendees and still I feel more secure about myself than when I am placed between my (or BF/GF) family. I never had a close connection to most of my family members. And I have learned (the hard way) that it's better to have a handful of friends whom you can trust and rely on for 200% then to have a huge family… Because I was too trustworthy, I am now more careful. Who to befriend, how to befriend and how far will the (friendship) relationship go? Those things keep me busy as well. I think it is not well, maybe professional is the best word to describe my feelings toward it, to send kisses to someone you barely know. Or hearts to friends who are in a relationship and "just" friends with you. It would feel weird to do so. It would feel unnatural. So when GF/BF would receive and/or send hearts without ever commenting that it's (here I go again) "unprofessional" (impolite or uncaring towards the loving relationship the receiver, and maybe the sender as well, is in). If someone would send me hearts, I would thank that person but also ask to send me some "Liefs" instead. I guess it would make me highly uncomfortable…
But still, when I notice such a comment somewhere, my head starts spinning in all the (wrong) directions. Too many bad experiences. And because it is hard for me to trust people, even the ones I love, it's hard to believe them as well as they come up with a lousy (to my eyes and mind) explanation. Also, if you wish to leave someone a comment on a public social page, keep it professional as well. Seeing that everyone with some internet experience can see your profile (when it's blocked, you simply create an account, ask to be friends… most people just click accept because that's the thing to do: collect "friends"…). When there are only weird messages and/or pictures… Well, when you go out to find a job, new employers will check those pages. First online impression is just as important nowadays. And so much more dangerous!
Back to the subject though, as I mentioned before (more than once I do believe), my mind tends to take me on a roller coaster ride through emotions when I am writing. And that can lead to total different subjects in my posts compared to what I started with. I don;t really mind, seeing if I have the need/urge to write about it, it must be important to me at that time. And that is, of course, what my blog site and journal are for: me. So I won't "warn" people anymore. Just read as long as you find it interesting. Comment if you feel like it, rate it if you think it sucks (or maybe you even liked it), and remember this is merely a place for me to vent. Anonymously and in public. Willing to share my thoughts, emotions, questions and not too often answers…
Can someone love too much? Am I capable of loving too much?
Do I let the people that I love and care about get away with too much?
Am I being used and just too blinded by my love to see it?
Am I making a fool of myself with all these silly doubts?
(why do these silly doubts hurt me..?)
Am I making my loved ones feel uncomfortable because I am such a scared "jealous" person?
Can't I really lose it because I never had it to begin with (following my previous blog post)?
Do I still have it but am I too stupid and blind to see? Or maybe too preoccupied with silly, scary, tearing thoughst?
Thanks all for reading. If you feel like it, comment and/or rate this post. I will NOT allow any span, so save you and me the time and just don't spam here, thanks!
As a dear friend always used to say: "We think positive."


text:
Active Tags
Insight • 08 November 2019The Brexit decision deadline of 31 October 2019 has come and gone. The European Union has granted the United Kingdom a time extension due to expire next year, on 31...
Case Study • 08 October 2019In 2014, Cynata Therapeutics commenced technology transfer and product development, and simultaneously commenced consultation with regulatory authorities regarding the approval pathways for its cellular therapy products.
Insight • 02 October 2019The cost of protecting innovations can be onerous. There are drafting costs, international filing costs, prosecution costs, and then they hit you with renewal fees every year.
Insight • 27 September 2019The WayBack Machine takes its name from a fictional time travel machine in the cartoon The Rocky and Bullwinkle Show.
Insight • 18 September 2019The World Intellectual Property Review reports that one Chinese practitioner estimated bad faith applications may account for as many as one in three trade mark applications filed in China.
Insight • 05 September 2019Whilst a secret invention may appear to be the antithesis of the intended function of our patent system, there are some instances where the publication of a patent application is...


text:
Selecting The Most Fitting Travel Insurance coverage By Matching Up Your Options
19 Jan
If you want to get the finest worth for your funds then travel insurance comparison can assist you select amid several insurance firms. It is feasible for companies to offer you premiums over the other depending on the variety of coverage you want not to mention the variety of insurance you are searching for. You can get a headache if you study the fine print of every single single policy and it is feasible that you will be deciding on one particular of them due to the fact you received tired of studying the policy and listening to its agents.
In this case, travel insurance comparison matrices normally consist of items like theft and loss of valuables, health-related support and evacuation, risks of flight cancellations or delays, emergencies and accidents, and the expense of attaining the insurance policy. In a position to cover these are diverse types of policies and some of them also have other contingencies. Other than identifying the kind of insurance coverage you want, you also have to consider about your price range.
There are various sorts of travel holiday insurance and these are single trip or multitrip travel, healthcare travel, enterprise travel, and the extensive variety. In this scenario, variations can be witnessed in their specs. Despite the fact that there are companies who are prepared to customize their insurance ideas for you, this means that the charges will go up too.
You may well get sick throughout a trip and this is where healthcare travel insurance will cover your health-related and dental wants including the medication and hospital expenses. You might be on a trip to some remote location in which a health care facility is miles away and this can be extremely useful contemplating that you might fall ill creating you to miss your scheduled trip back. In terms of what medical insurance can also cover, these are preferences for a hospital or clinic not to mention the preference for a variety of medical access or tools.
When it comes to single trip insurance, this is legitimate for a single trip. Even if it is good for a single use only, it can be renewed and is extensive and covers most of the risk factors described. In scenario that you are organizing for that prolonged awaited dream trip then this is the insurance you require.
The next level in our travel insurance comparison is the multitrip or annual kind. An extension of the singletrip is what this is and regardless of how much you travel the coverage is good for 1 year. Right here, upon expiry, you can renew it but it wants for you to pay the premium expense yet again and it raises each and every year to accommodate fiscal fluctuations such as inflation charge and tax.
Organization travel insurance, on the other hand is much far more favorable to these who go on a very short outofcountry trips. A brief trip would final as tiny as a few to four days. The important thing about this sort is the cruise or flight worries this kind of that any delays or cancellations can have a large effect on the travel schedule.
With the inputs in the travel insurance coverage comparison table, complete insurance costs the most. Here, these could be catered by a distinct type of insurance coverage policy and may possibly carry unnecessary provisions. It is essential that you contemplate all your alternatives very first before agreeing to any policy.
Get to know the distinct types of insurance offered and then proceed to an appleistoapple comparison which is the next stage. You need to have to give a higher rank to the healthcare coverage or any other threat element that you want to be able to locate the greatest policy. Preserve in mind that disregarding individuals that did not meet your necessity will support you find the greatest options to talk about with your agent.
Deciding about insurance coverage can be easier if you make use of travel insurance coverage comparison. You need to have not fear about issues when you have insurance. This way, you can take pleasure in your muchneeded vacation in the most protected and hassle-free way.


text:
Chiropractic and Canada
The word of the day for the chiropractic profession, every day, for the last 124 years, has been
schismatic: characterized by or favoring schism., a split or division between strongly opposed sections or parties, caused by differences in opinion or belief*.
On one side of the schism, the opinion is that chiropractic care is a safe, low cost, and effective method of alleviating neck and back pain. On the other side of the schism, the opinion is that chiropractic care is effective for pain relief and also affects the function of the nervous system in ways that are only just beginning to be fully understood and provide new insight into well over a hundred years' worth of clinically observed, documented, and sometimes dramatic improvement to a surprising variety of physical functions.
In spite of a burgeoning body of scientific evidence that supports over a century's worth of anecdotal evidence showing that adjusting subluxations improved patients' physical and neurological functioning, a vocal minority of chiropractors would like to limit the scope of chiropractic practice solely to the treatment of neck and back pain. And then what?
Take eight minutes to hear Dr. Rob Scott, president of Life University, share his alarm that this dangerously limited view of chiropractic is an "intellectually dishonest and misinformed approach to the third largest portal of entry, and I might emphasize, drug-free healthcare profession on the continent…"
To allow those narrow, misinformed interests to achieve regulatory agency over chiropractic in Canada – or anywhere else in the world – serves neither our principles nor the future of our profession.
Scroll down to find a transcript of Dr. Scott's remarks.
Chiropractic and Canada, Transcript
"Hey, everyone. Dr. Rob Scott here.
You know, there's been a situation occurring in several countries, but most recently in my homeland of Canada, about chiropractic that is being driven by a very small, narrow, politically focused segment of the profession, but it's resulting in rapid regulatory changes to the profession all across the country. Regulatory changes that are troubling because they're based upon misinformation, innuendo, and a frank bias. These regulatory changes that are occurring are significantly limiting what practicing chiropractors are legally permitted to say and do regarding the benefits of chiropractic, the sorts of benefits that for over 124 years, those who have practiced in this profession have witnessed daily in their offices. I'll say that as the president of Life University, the largest vitalistic university in the world, this is personally frustrating. You see, Life University is a fully accredited institution in Marietta, Georgia that offers 24 degree programs at the associate's, bachelor's, master's, and the professional Doctor of Chiropractic degree level, and our College of Chiropractic is the largest single campus chiropractic college in the world.
And as per our mission, all of our academic programs, be it nutrition, dietetics, exercise science, positive psychology, environmental studies, et cetera, and chiropractic, are based upon a vitalistic philosophy, which simply put is the belief that living organisms are fundamentally different from nonliving things because they're governed by different principles than are inanimate objects such as rocks. It's not really a difficult concept to grasp. Contemporary vitalism, or more accurately neovitalism, recognizes that all living systems are biologically intelligent, self-organizing, self-maintaining and self-healing, and express these attributes when the system is free of interference. For example, the environment would flourish if free of the interference of people; people flourish when free of the interference from toxins that we breathe in and ingest daily; and free from the interference and destruction of negative thoughts and emotions. For chiropractors who look at health through a vitalistic lens, their objective is to identify and remove interference to our lives, specifically to nerve system function through correction of verbal subluxation.
I say I'm frustrated because once again across Canada, a political narrative that vitalists and subluxation [are] anti scientific, dogmatic and not evidence-based has been put into play by those who outright reject or simply do not desire to understand the position.
At a time when the entire human physical experience is being corporately exploited for the singular purpose of selling the consumer more drugs, and when the US is in the midst of the single biggest drug addiction epidemic in our history, I'm frankly alarmed that such an intellectually dishonest and misinformed approach to the third largest portal of entry, and I might emphasize, drug-free healthcare profession on the continent is being undertaken.
Rob Scott, D.C., Ph.D.
Now, I'm certainly aware that on occasion some members of the profession make rather extreme claims that don't benefit either the profession and certainly the public, and those individual cases should be addressed by regulatory boards. But one has to acknowledge that even those claims do not require the vast list of potential side effects, ranging from dizziness and nausea to death, that the medically approved evidence-based drug companies must add breathlessly and in fine print to their advertisements. And while there's still certainly debate within the profession amongst those who embrace a vitalistic view as to the operational and scientific understanding of the word subluxation, I would think that for the sake of ease, our Canadian colleagues and certainly those in Ontario could simply look to the Ontario Regulated Health Professions Act of 1991 itself. In the Chiropractic Act, Chapter 21 Section 4.1.i, it states …, "a disorder arising from the structures or functions of the spine and their effects on the nervous system."
In short, one could ascertain that the term subluxation refers to a collection of clinical findings having to do with spinal malfunction, muscle and soft tissue abnormalities, neurological alterations, and their impact on movement and function, all of which by the way are well documented. As to the false assertion that subluxation-based chiropractic is anti-scientific and not evidence-based, the question in their minds must come down to: What is one willing to accept as evidence?
You see, there's plenty of research available, small clinical trials and case reviews that support our vitalistic premise, and certainly more good work is required in this area. However, it appears that from our critics' perspective, the availability of high-level systematic reviews and randomized clinical trials are to be the onlyacceptable metric for research in general and chiropractic care specifically, once again, driving their singular agenda. But you know, one might ask hockey superstar, Sidney Crosby, what he thought of the groundbreaking chiropractic care he received at our university that was featured in Sports Illustratedand Maclean'smagazines, [and] that put him back on the ice in 2011 after concussion threatened to end his career.
That, of course, is the anecdotal evidence that our critics outright and flatly reject. Once again, refusing to acknowledge that "ask the man who's been there" is folk wisdom that's still highly relevant in the clinical encounter. You see, evidence-based practice is determined by three factors, patient preference, best available evidence, and the provider's clinical experience, not simply evidence. So while subluxation-based chiropractic care may not be the conventional approach to health that people seek, to suggest that there's no evidence for this is simply disingenuous and inaccurate.
It appears that, once again in Canada and alas, my home province of Ontario, a politically biased agenda has been launched by the detractors of our vitalistic, subluxation-based view of healthcare, and the drug-free natural lifestyle that it invokes. And, as per their usual and customary playbook, personal attacks have been launched against individuals who support the vitalistic view and have influence in provincial regulation, all in an effort to discredit them and have them removed from their positions of influence on regulatory boards.
Because of that, I encourage all chiropractors, across Canada and other regions around the world who feel that their principled chiropractic approaches under attack, to hold their associations, their regulatory boards, and their leadership accountable to represent all chiropractors, not just a few vocal ones who see the world and the practice of chiropractic in their likeness.
Rob Scott, DC, PhD
So while the consumer continues to demand chiropractic care, I'm going to leave this renewed debate in the hands of the practicing chiropractors in Canada, and the provincial associations and regulators, taking comfort in knowing that every day thousands of people seek effective chiropractic care for a variety of ailments and leave their chiropractor's office satisfied, feeling better, and more able to engage in and enjoy the day that lies ahead.
Those are just a few of my thoughts and I hope you'll join us in Montreal at LIFE Vision Canada this March 29th and 30th or down here in Atlanta for LIFE Vision in April on April 12th and 13th. information for both events can be found at lifevisionseminars.com.
Thanks for tuning in. We'll see you next time."
Download a PDF of the transcript here: 190306 Scott transcript Chiropractic and Canada
*Definitions of schism and schismatic are courtesy of Google Dictionary.
Links to material referenced in Dr. Scott's remarks: https://www.ontario.ca/laws/statute/91r18
Latest posts by TCL Team (see all)
- Rubicon in Paris - August 11, 2019
- A Dangerous Blast From the Past - June 17, 2019
- The Sublime and the Dangerous - June 17, 2019


text:
Unsure about Dapple Designs and whether we can deliver the level of professionalism you require for your landscape design needs? View our sample plans above to see what we offer and what you will be paying for.
We offer a comprehensive range of services that can be tailored to your requirements and budget.


text:
AHIMA, IHE Seeking Public Comment on Patient Registration White Paper
Integrating the Healthcare Enterprise (IHE) and the American Health Information Management Association (AHIMA) are seeking public comments on their joint white paper entitled "Patient Registration Demographic Data Capture and Exchange."
Based on the AHIMA Patient Registration Use Case, the paper was developed by the IHE Patient Care Coordination (PCC) Committee with participation of the AHIMA Standards Task Force. The paper specifies the requirements and constraints for patient demographic data that should be collected and exchanged for patient registration during an emergency department visit at a healthcare provider.
"The goal of IHE is to promote implementation of standards-based solutions to improve workflow and access to information in support of optimal patient care," the authors wrote. The processes outlined in this white paper contribute to the growing body of work in developing standards for HIM practices.
The intended audience of the white paper includes healthcare IT departments, technical staff of vendors participating in the IHE initiative, experts involved in standards development, and individuals interested or involved in integrating healthcare information systems and workflows, according to the white paper.
A press release stated that these data requirements "may be proposed to be published as future IHE Technical Framework Volume 4: United States National Extension to the IHE ITI Patient Administration Management (PAM) Profile for the message-based data exchange."
The white paper is available for download here. Those who wish to submit comments may download the Public Comment Form here. The form contains instructions for recording and submitting comments.
Sarah Sheber (firstname.lastname@example.com) is assistant editor and web editor at Journal of AHIMA.


text:
Raipur (Chhattisgarh)[India], Oct 16 (ANI): Chhattisgarh Chief Minister Raman Singh on Sunday inaugurated Deendayal Upadhyaya auditorium in Raipur on the occasion of his birthday.
Speaking at the event, Singh said the auditorium has been named after Pandit Deendayal Upadhyay so that all can get inspired by his illustrious life.
The Chief Minister further said the auditorium has become state's pride as it was constructed in just two years.
"The people of Chhattisgarh have created me and I am trying to live up to their expectations. The journey from councilor to Chief Minister was possible only because of people's blessing," he said.
Singh further called for bringing quality in work and asserted that it would help the Bharatiya Janata Party (BJP) to earn people's confidence.
"I have always pitched for sound and quality work so that the coming generation would remember our work. If we will do quality work then nobody can beat BJP till generations," he added.(ANI)


text:
100% Completion Guide for Resident Evil 2 Remake.
Other RE2 Guides:
- All Endings.
- Beginner's Guide.
- All Mr Raccoons Locations.
- Lockers, Safe Codes / Combinations and Leon's Desk.
- The Tofu Survivor Bonus Mode (Characters / Unlocks).
- Weapon's Upgrades Location.
- Laboratory Puzzle Solution.
- Items / Keys Location.
- Weapon's Guide.
Puzzle Solutions
Click to enlarge...
Story Related
The achievements mentioned below are all story related so they can't be missed.
Welcome to the city of the Dead
Make it to the police station. This achievement is unlocked right after you exit the gas station, just keep playing the game and rejoice your first one!
Path to the Goddess
Solve the Goddess Statue puzzle. This one requires you to get all 3 medallions and insert them at the statue located at the police station.
Never-Ending Rain
Escape the police station. You get this achievement after you go through the parking garage on to the "Behind the RPD" section.
A Great Need for a Shower
Escape from the sewers. After the fight with 2G (William Birkin First appearence) your will be granted this achievement.
Endings and Difficulty
Achievements that you get by beating the game or certain stages several ways.
A Hero Emerges
Complete Leon's story. Beating the A or B campaign with Leon unlocks this.
A Heroine Emerges
Complete Claire's story. Beating the A or B campaign with Claire unlocks this.
Hack Complete
Complete Ada's segment. During Leon's campaign you get to control Ada, beating this segment unlocks this. Don't forget that you can also get the achievement called "One Slick Super-Spy" while you play with Ada. For more information on how to get that achievement see the Misc section.
Hide and Seek
Complete Sherry's segment. During Leon's campaign you get to control Sherry, beating this segment unlocks this. Don't forget that you can also get the achievement called "Youg Escape" while you play with Sherry. For more information on how to get that achievement see the Misc section.
Broken Umbrella
Witness the true ending. You get this achievement when you beat any B scenario. To unlock the ability to play on a B scenario you must play with Leon or Claire for the first time.
Leon "S." Kennedy
Complete Leon's story on "Standard" or "Hardcore" with an S rank. A S rank is get by beating the game within 3:30 hours in case you are playing an A scenario, or 3 hours in case you are playing a B scenario. That is true for the Standard difficulty because if you are crazy enough to do it on Hardcore mode, then the times are 2:30 hours for the A scenario and only 2 hours for the B one. Therefore the easiest way to do it is on Standard mode scenario A for Leon.
Sizzling Scarlet hero
Complete Claire's story on "Standard" or "Hardcore" with an S rank. A S rank is get by beating the game within 3:30 hours in case you are playing an A scenario, or 3 hours in case you are playing a B scenario. That is true for the Standard difficulty because if you are crazy enough to do it on Hardcore mode, then the times are 2:30 hours for the A scenario and only 2 hours for the B one. Therefore the easiest way to do it is on Standard mode scenario A for Claire.
Grim Reaper
Complete "The 4th Survivor" extra mode. It unlocks when you beat a B scenario for Leon or Claire.
Hardcore Rookie
Complete Leon's story on "Hardcore" game mode.
Hardcore College Student
Complete Claire's story on "Hardcore" game mode.
Complete the game without using a recovery item. It is the easiest done in Assisted difficulty where the enemies are a little weaker and you recover a certain amount of health automatically.
Clear the game without opening the item box. Best done in Assisted difficulty where the enemies are a little weaker and you recover a certain amount of health automatically. If you pick up all of the Hip Pouches along the way that will help you a lot. Also if you unlocked any of the infinite weapons it will make your life easier.
A Small Carbon Footprint
Take 14000 steps or fewer in one playthrough. Doing a speedrun and getting a S rank of the game will naturally unlock this.
Bosses and Enemies
This section is dedicated to different ways to "play" with the zombies.
Don't Need No Stinkin' Gun
Defeat an enemy with a knife. Don't need to be all knife hits to get this one, so you can shoot a zombie down to the floor and then hit him with the knife until you kill it.
Bon Appétit
Shoot the grenade you fed to an enemy. Equip a grenade as a defense item, then let a zombie grab you and press space to use it. Then step back and quickly shoot it to send him your regards and get the achievement.
Zombie Roundup
Kill 3 enemies at once with a sub-weapon. A good place to do this is in Jail with Leon when the cells are released. Just herd them and throw a grenade to get the achievement.
Like Skeet Shooting
Shoot a zombie dog or a licker out of the air. Best done with Leon's shotgun when you first encounter a zombie dog at the Parking Garage.
Keep Their Heads Ringin'
Paralyze a licker's sense of hearing. When you first encounter a licker use a flash grenade on him to stagger it and get the achievement.
Hats Off!
Shoot Tyrant's hat off his head. Just a single pistol shot will do, aim for his head and the achievement is yours.
Defeat Stage 2 G using the crane only once. The fight against him is at the final part of the Sewers. Shoot him down 2 or 3 times to make sure that the first crane use is also his last. If he gets knocked down that means you got the achievement.
With Time to Spare
Defeat Stage 4 G with 4+ minutes left until detonation. This is in Claire's campaign, kill William Birkin as fast as possible outside the train car.
In The Blink of an Eye
Defeat Super Tyrant with 5+ minutes left until detonation. This is in Leon's campaign Mr. X in his final form will come at you, just down him as fast as possible. After receiving enough damage Ada will send you a rocket launcher to finish him off.
All the collectibles in the game can be found here.
Vermin Extermination
Destroy a Mr. Raccoon. First, go to the S.T.A.R.S. office like below picture:
Treasure Hunter
Using the photo hints, find 2 hidden items. First you need to get to the Workroom in the Sewers and get the roll film called "Hiding Place". Then you must head to the Dark Room back in the Police Station. Do not worry you will find a shortcut to it around the room where you found the roll film, assuming you have already the T-Bar Valve Handle. Once you reach it, reveal the film to get 2 pictures containing different places for you to loot, being those the Press Room, and the STARS Office. Do a quick search of the desks and you should get your rewards as well as this achievement.
Lore Explorer
Read all of the files.
Complete Vermin Extermination
Destroy all Mr. Raccoons. Read this guide.
Everything else that doesn't fit into the other categories.
The Basics of Survival
Combine two items together. You can get this several ways like combining green herbs with red ones, or greens with greens, but interestingly most likely you will get it first by modding your pistol. See the achievement "Customizer" for more details.
Hip to Add Squares
Increase your inventory slots. Done after you open the dial safe located at the West Office 1F that will provide you with the item needed. The code for it is as follows: 9left - 15right - 7left.
Also, You can increase your inventory slots by finding bag in below place:
As well, you can get next bag in below locker (code: NED (left) and MRG (right):
Customize a weapon. Obtainable after you open the "chest" locked by 2 dial locks at the West Office where the police is throwing a welcoming party dedicated to Leon. The code for the left lock is "NED"; the one for the right lock is "MRG". After that combine the item with your pistol.
Eat This!
Counterattack with a sub-weapon. After you get your first defense item, let a zombie grab you and press space when the time is right to use it.
That'll Hold 'Em
Use Wooden Boards to board up a window. After you go through the Reception you will encounter a zombie banging on the window. Pick up some Wooden Boards just to your right and use them there to get the achievement.
A Vault-like Mind
Open a portable safe.
Probably everyone has a different combination. If you have portable safe in your equip, click on buttons in correct sequence. Lights must to turn on green in the order shown in the picture below. Check which button lights which lamp (I prefer write it on the sheet of paper), then click on it in the correct order:
First Break-in
Open a dial safe. Gettable after you open any of the ones avaible throughout the game like the one at West Office. See "Hip to Add Squares" achievement for more information.
A Waist of Space
Expand inventory slots to max.
One Slick Super-spy
Use only the EMF Visualizer to complete Ada's gameplay segment. Do not use the pistol or any sub weapons you may get or else you will fail to get the achievement.
Young Escape
Escape the bedroom within 60 seconds during Sherry's segment. Pick up the Stuffed Doll on the chair. Then examine it on your inventory, turn it arround and press Space. It will reveal a block that you need to insert at a puzzle near the door. Solve it and get the Scissors needed to cut yourself out of the bedroom.
Master of Unlocking
Open all of the safes and locks in the game. See the "Tips" section of this guide for more information, where you will find all the safe and lock codes there are.
The Ghost Survivors DLC
Hell of a Sheriff
Complete the No Way Out scenario (No training mode). You unlock this mode when you complete all of the 3 previous ones: No Time To Mourn, Runaway and Forgotten Soldier. Watch the video below for a playthrough.
Got 'Em
Destroy all Mr. Raccoons hidden in The Ghost Survivors mode. Watch the video below for their locations.


text:
Joah Love Linen Aurora Heart Girls Top
$8.99 $36.00
Free U.S. Shipping on all orders $99+
Have a question? 800.393.5840
In a pretty shade of linen with grey tiny hearts printed all over it, this tee by Joah Love is subtle but special. It has an asymmetrical hemline with a tie on the side for extra flair. Pair it with the matching leggings, or with any of her denim shorts. It has a slightly boxy cut that allows for free movement and keeps her cool (in more ways than one!)


text:
There are concerns that imidacloprid-based insecticides for thrips control may negatively affect Rhizobia in inoculants when tank-mixed and applied in-furrow when plating peanuts.
As we are just a week (or less) from starting peanut planting in Virginia, I thought this issue should be addressed for farmers' peace of mind if they decide to mix Optimize Lift inoculant with Admire-Pro insecticide for in-furrow seed treatment. I personally have not looked into this issue before, because we have not seen any effects in research plots or received complaints from farmers. However, we will research this aspect in 2018.
Meanwhile, my colleague Dan Anco, Peanut Specialist at Clemson University, already tested the effect of different tank-mixtures for in-furrow applications on the nodule number and peanut yield in 2015 and 2017. Graciously, Dan has shared this information included in the In furrow tank mixes tables.
The results clearly show no effect of mixing Optimize Lift with Admire-Pro on peanut yield in both years (Tables 1 and 2), even when the number of nodules from this mixture appeared to be reduced by the use of Admire-Pro in 2017 (Table 2). David Jordan, Peanut Specialist at North Carolina State University, reported similar findings: Peanut Science Inoculant CFTGM Peanut Inoculant.


text:
Keep your property and your employees protected with business insurance in Cambridge, from
Commercial insurance is an involved concept with a variety of aspects and variables to think about. In comparison to other insurance products, it's not as easy to compare quotes amongst providers online. So which insurer can you trust?
Business insurance in Cambridge from
When it comes to home-business insurance in Cambridge, the process can be as simple as talking to your Insurance Advisor about a policy extension, especially if you currently have an existing home owner's policy.
Feel free to contact an Insurance Advisor, who will be more than happy to explain all of the details, answer any questions you might have, and let you know that
As a responsible corporate citizen, we believe in balancing our economic, environmental and social priorities. In fact,
Cambridge has a public business policy system that provides a minimum level of mandatory coverage to vehicle owners when they purchase license plates.


text:
- Sleeps 5 - 8
- Length 12' 4" - 18' 7"
- Weight 2250 - 3375 lbs
Expandable Trailer
Find an Expandable Trailer
From pop-ups to slide-outs, expandable trailers encompass any RV that allows you to add extra living space once you're parked. Expandable sections can be hard-sided slide-outs in the bedroom or living space, soft-sided pop-out sections, or tents that let you enjoy nature while protecting you from the elements. Expandable trailers are about flexibility and choice, both on the road and in camp.


text:
Scale: 1:72
Kit type: Injection moulded Decal options: (four) No. 5, 12 Grupo, 1937, No. 46, Barajas, 1938 and No. 1, Grupo 24, 1937, all Spanish Republican air force; and 20W+I, Spanish Nationalist air force, Logrono, 1940 UK price: £10.99
When the Tupolev SB 2 first flew in 1934 it marked quite an advance, not just in Soviet, but in world aircraft design. Its mid-wing monoplane layout, smooth alloy skin and retractable undercarriage all added up the justify the aircraft its initials Sb – Skorstnoy Bombardirovshchik, or HighSpeed Bomber. By 1937 the first production aircraft fitted with the M-100 engines and capable of achieving 263 mph (423 km/h) were entering service. The SB 2 made its combat debut during the Spanish Civil War. Operated by the Republicans it was nicknamed 'Katiushka' and 'Martin Bomber' by the Nationalist forces.
The product reviewed here is one of three versions of this kit released to date by the Ukrainian company ICM. It contains parts and decals for four Spanish options for both the Republican and Nationalist forces. It represents a welcome choice of subject by ICM since previous kits by FROG and MPM both have issues with quality and accuracy. The ICM kit comprises a surprising 164 parts on six grey plastic sprues. Surface detail is comprehensive and very fine, although a little flash is present on some parts. The clear sprue has several different canopy options and is thin and acceptably transparent. The small decal sheet carries four options, three Spanish Republican and one captured Nationalist aircraft. The instructions give clear and precise assembly diagrams and reference is made to Model Master paints for cockpit colours, etc. Before any assembly can begin excessive mould release oil has to be washed from all the plastic items.
Surprisingly for such a small aircraft, the fuselage is broken down into three sections. This, I believe, is due to moulding limitations. The wing centresection assembles first. The open bomb bay is well detailed and several bomb load options are provided. There is also plenty of internal structure provided in the undercarriage bays. The nose compartment for the navigators is well furnished and fits nicely onto the completed centresection, as does the rear fuselage. The rear gunner's position is very well detailed. The outer wings are assembled next and these slot onto spars which give a good joint. The last major items are the engines. Care is required to achieve a decent fit with these since each comprises six parts and they must be correctly aligned or you will have problems attaching them to the wings. All the control surfaces are separate so that you can offset them if desired. Finally, the transparencies were fitted and the model was prepared for painting. I substituted Xtracolor and Humbrol paints for those given in the detailed camouflage guide. Markings are provided for the three Republican aircraft in green and brown mottled camouflage with red identification bands, green and brown disruptive camouflage and sandy brown upper camouflage, respectively, and for the Nationalist machine in green, brown and grey splinter camouflage.
I enjoyed making this model. It is accurate, the level of detail is very good and if care is taken the many sub-assemblies come together to give a nice representation of the SB 2. This is another good offering from ICM and I hope more versions will follow in the future.
Connected themes: large scale aircraft models, concorde model plane, hobby shops, SB 2M-100 'Katiushka' Spanish Air Force Bomber, how to build aircraft, model plane plans free, rc airplane kits.
Comments are closed.
The website contains material from different sources. Content on the website is provided for informational purposes. All trademarks mentioned in the website belongs to their owners or companies.