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The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.
Materials and Methods
Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.
Principal Findings
Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).
Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.
Author Summary
Plasmodium vivax is responsible for approximately 60–80% of the malaria cases in the world, and contributes to significant social and economic instability in the developing countries of Latin America and Asia. The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. The innate immune receptors recognize Plasmodium sp. and initiate a broad spectrum of host defense mechanisms that mediate resistance to infection. However, the innate immune response is the classic "two-edged sword", and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α during acute malaria. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Unexpectedly, we noticed an impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. We proposed that neutrophil paralysis is in part responsible for the enhanced susceptibility to bacterial infection observed in malaria patients.
Citation: Leoratti FMdS, Trevelin SC, Cunha FQ, Rocha BC, Costa PAC, et al. (2012) Neutrophil Paralysis in Plasmodium vivax Malaria. PLoS Negl Trop Dis 6(6): e1710. doi:10.1371/journal.pntd.0001710
Editor: Mauricio Martins Rodrigues, Federal University of São Paulo, Brazil
Received: February 17, 2012; Accepted: May 13, 2012; Published: June 26, 2012
Copyright: © 2012 Leoratti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (R21 AI080907 and RO1 AI079293) to RTG and DTG, Malaria Network (CNPq-555646/2009-2/FAPEMIG/MS-CBB-APQ-01153-10),National Institute of Science and Technology for Vaccines (CNPq-573547/2008-4/FAPEMIG/MS-CBB-APQ 00077-09), and the Program for Technological Development in Tools for Health from Fundação Oswaldo Cruz (PDTIS-FIOCRUZ- RVR22). RTG, FQC and LRVA are research fellows from CNPq; DTG and FMSL are research fellows from FAPEMIG and FIOCRUZ, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Malaria is a complex disease that affects approximately 300 million people every year. Among the different Plasmodium species that infect humans, P. falciparum is the main cause of deaths in sub- Saharan Africa. On the other hand, P. vivax is responsible for approximately 60–80% of the malaria cases in the world [1], [2], and contributes to significant political, social and economic instability in the developing countries of Latin America and Asia [1].
The innate immune system recognizes Plasmodium sp. by different pattern-recognition receptors and initiates a broad spectrum of defense mechanisms that mediate host resistance to infection [3], [4]. However, the innate immune response is the classic "two-edged sword", and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. The outcome of infection depends on a balance between pro- and anti-inflammatory responses allowing the formation of an effective immune response, while limiting its pathogenic potential [5], [6], [7].
Toll like receptors (TLR) play an important role in recognition of pathogens through distinct pathogen-associated molecular patterns (PAMPs). Activation of TLR on monocytes, dendritic cells and neutrophils can induce changes in the expression of surface proteins and release inflammatory mediators such as cytokines and chemokines. The production of cytokines amplifies innate immune responses and shapes the development of acquired immunity. In addition, activated myeloid cells release high levels of reactive oxygen species (ROS) and antimicrobial peptides that efficiently kill invading pathogens [8], [9].
It is noteworthy that glycosylphosphatidylinositol anchors and DNA from Plasmodium parasites are important PAMPs that activate TLR during malaria [10], [11], [12], [13]. Some in vitro studies show that phagocytosis of opsonized hemozoin (HZ) decreases expression of HLA-DR in monocytes [14], [15]. On the other hand, study has demonstrated that DNA bound to HZ induces monocytes to produce high levels of cytokines and contribute to dendritic cell maturation [16]. While other report evaluated activation of polymorphonuclear cells and observed elevated levels of myeloperoxidase, lysozyme and lipocalin in patients with severe malaria [17], the involvement of neutrophils in malaria pathogenesis has been poorly investigated.
Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α in response to TLR agonists during acute malaria and seem to be the main source of pro-inflammatory cytokines in the blood. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Interestingly, we noticed an enhanced expression of G-protein receptor protein kinase (GRK2) associated with decreased levels of CXCR2 and impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. Our findings indicate a mechanism by which malaria patients may become more susceptible to bacterial infection.
Ethics statement
All protocols and consent forms were approved by the Institutional Research Board from University of Massachusetts Medical School (IRB-UMMS 10268), the Ethical Committees on Human Experimentation from Centro de Pesquisa em Medicina Tropical (CEP-CEPEM 095/2009) and Centro de Pesquisas René Rachou – Fundação Oswaldo Cruz (CEP-CPqRR 2004), as well as by the National Ethical Committee (CONEP 15652) from Ministry of Health, Brazil. A signed informed consent was obtained from each subject prior to enrollment in the study.
Patients were recruited and examined at CEPEM in Porto Velho, a malaria endemic area in the Amazon region of Brazil. Up to 100 ml of peripheral blood was collected immediately after confirmation of P. vivax infection by thick blood smear film and 30–45 days after chemotherapy (n = 26, ranging from 18 to 66 years old [35±9.5]). Patients were treated for 10 days with chloroquine and primaquine. P. vivax infection was confirmed by polymerase chain reaction (PCR) analysis [18]. The clinical manifestations of malaria were fever, myalgia, chills, arthralgia, nausea, vomiting or diarrhea, but no patient had complicated malaria. Peripheral blood was also collected from 15 healthy donors (HD) ranging from 21 to 56 years old [32±8] living Porto Velho and negative for P. vivax infection by thick blood film and PCR. All the experiments were done using fresh cells.
Cellular immunophenotyping
Whole blood was stained with antibodies from ebioscience: anti-CD14-FITC (clone 61D3), anti-TLR2-PE (clone T2.5), anti-TLR4-PE (clone HTA125), anti-HLA-DR-PE (clone L243), anti-CD14-APC (clone 61D3), anti-CD62L-APC (clone DREG56); BD Bioscience-Pharmingen: anti-CD66b-FITC (clone G10F5), anti-CD88-PE (clone C85-4124), CXCR2-PE (clone 6C6), anti-CD16-PercpCy5.5 (clone3G8), anti-CD15-FITC (clone HI98); R&D: CCR2-PE (clone 48607) Subsequently, red blood cells (RBC) were lysed with FACS lysing solution (BD Biosciences) following manufacturer's instructions, washed with phosphate buffered saline (PBS) and maintained in paraformaldehyde 2% (PFA) until the acquisition on an FACScan upgraded with a second laser (5 colors). The software used for acquisition was CellQuest Pro from BD and Rainbow from Cytek. Data analyzed using FlowJo version 9.3.2 (TreeStar)
Purification of monocytes and neutrophils
Peripheral blood mononuclear cells (PBMC) and neutrophils were enriched on-site by gradient centrifugation over Ficoll-Paque™ plus (GE-Healthcare). Isolated PBMC were washed twice in RPMI, ressuspended at 5×107/ml in PBS supplemented with 2% heat- inactivated fetal bovine serum (FBS) and 1 mM EDTA. Monocytes were purified from PBMC with the Human Monocyte Enrichment Kit (STEMCELL-Technologies) following manufacturer's instructions. The cell layer containing the granulocytes were collected just above the red blood cells phase and the cell suspension was lysed with NH4Cl 0.15 M with KHCO3 0.1 M and Na2EDTA 0.1 mM. Cells were ressuspended in PBS (2% FBS, 1 mM EDTA) at final concentration 5×107/ml. Purification of neutrophils were performed with the Human Neutrophil Enrichment Kit (STEMCELL-Technologies) following manufacturer's instructions. Purity of monocytes and neutrophils was measured by flow cytometry after staining with mAbs specific for CD14, CD66b and CD16. Purified monocytes were CD14+CD16−CD66b− and neutrophils were CD14−CD16+CD66b+. Cell preparations reached over 98% of purity and viability of 90–100%.
Tissue culture assays
Purified monocytes and neutrophils were plated in 96-well cell culture plates at a final concentration 2×105/well in RPMI1640 (Sigma Aldrich R6504) (10% FBS and 100 µg/ml streptomycin/100 U/ml penicillin) in the presence or absence of Toll like receptors agonists: 100 ng/ml lipopolysaccharide (LPS - TLR4 agonist, Sigma-Aldrich) or 100 ng/ml Pam2CSK4 (Pam - TLR2/TLR1 agonist, InvivoGen). Supernatants were harvested after 48 hours of culture and kept at −20°C until cytokine measurement.
Cytokine measurements
IL-8 (CXCL8), IL-1β, IL-6, IL-10 and TNF-α were quantified in cryopreserved plasma and supernatants from monocyte and neutrophil cultures using the Cytometric Bead Array kit (CBA, BD Biosciences Pharmingen) following manufacturer's instructions. The limits for cytokine detection were 3.6 pg/ml (IL-8), 7.2 pg/ml (IL-1β, 2.5 pg/ml (IL-6), 3.3 pg/ml (IL-10) and 3.7 pg/ml (TNF-α).
Phagocytosis and superoxide assays
Phagocytosis was assessed by incubating neutrophils for 20 min with opsonized zymosan (5×106 particles/cell) in a glass slide. Cells were stained by the May-Grünwald-Giemsa (Rosenfeld) method (Laborclin Pinhais, PR, BR). The number of cells containing phagocytic vacuole was determined by counting 100 neutrophils. Nitro Blue Tetrazolium (NBT) assay was performed using peripheral blood. Briefly, 25 µl of blood sample were added to the same volume of NBT (1 mg/ml) at 37°C for 15 min and an additional 15 min at room temperature. Percentage of cells reducing NBT was established by counting 100 neutrophils.
Chemotaxis assay
Chemotaxis was performed with purified neutrophils using a 48-well microchamber (NeuroProbe, Gaithersburg, MD) with a 5 µm pore polycarbonate membrane. Fifty thousand neutrophils were allowed to migrate toward IL-8 (CXCL8) (10 ng/ml), CCL2 (1 ng/ml), or medium alone. After 1 h incubation, at 37°C with 5% CO2 the membrane was removed, fixed and stained. The number of neutrophils that migrated through the membrane was counted under a light microscope on at least 5 randomly selected fields.
Real-Time PCR
Total RNA from neutrophils was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA to cDNA was carried out by reverse transcription (Superscript II, Gibco Life Technologies Grand Island, NY). Real Time PCR (qPCR) analysis of CXCR2 receptor mRNA profile was performed with the Platinum SYBR® Green qPCR SuperMix-UDG kit (Invitrogen-Life Technologies, USA) and the reactions were processed in ABI Prism 7500 equipment (Applied Biosystems, Warrington, UK) was performed in an ABI Prism 7500. The reactions were carried out in a final volume of 10 µl containing 6.25 µl of SYBR Green, 0.5 µM of each primer (designed on the basis of the mRNA sequence of the receptor gene available at the GenBank) and 1 µl of cDNA. RT-PCR cycles were 95°C for 15 s and 60°C for 1 minute and the dissociation curve was constructed by increasing temperatures from 60°C to 90°C. The primers used were: CXCR2 sense: 5′- AGATGCTGAGACATATGAATTT -3′ and CXCR2 antisense: 5′- CTTTTCTACTAGATGCCGC-3′, β-Actin sense: 5′-GCTCGTCGTCGACAACGGCTC -3′ and β-Actin antisense: 5′- CAAACATGATCTGGGTCATC -3′. The expression of β-actin mRNA was used as a control for all samples. The relative level of gene expression was determined by the comparative threshold cycle (Ct) method using the formula −2ΔΔCt whereby data for each sample were normalized to β-actin mRNA levels.
Immunofluorescence assay for GRK2
Neutrophils purified on glass slides were fixed with 4% paraformaldehyde in a wet chamber at room temperature. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (Amersham Pharmacia Biotech, CA) and blocked with 1% bovine serum albumin (Sigma-Aldrich) in PBS containing normal goat serum (1:50). Cells were then incubated overnight with polyclonal rabbit anti-GRK-2 antibody (1:200, clone C-15, Santa Cruz Biotechnology) followed by incubation with red fluorescent Alexa Fluor 594 (goat anti-rabbit IgG (H+L); 1:400, Life Technologies). Nuclear material was stained with 4, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Images of stained cells were captured using an epi-fluorescence microscope (BX-40; Olympus, Japan). The mean fluorescence intensity was determined from a linear measurement of fluorescence of individual cells using Image J software (Imaging process and analysis in Java, National Institute of Mental Health). Ten cells of five randomly chosen fields of each slide were analyzed.
Statistical analysis
Statistical analysis was performed using Prism software, version 5.0 (GraphPad). The results were analyzed using two-tailed paired test. Wilcoxon testing was used when data did not fit a Gaussian distribution. The results were analyzed using unpaired t test when two groups were compared. Mann-Whitney (MW) test was used when a normality test failed. Differences were considered to statistically significant, when p≤0.05.
High levels of circulatory cytokines in P. vivax malaria patients
High levels of the pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were found when comparing the sera from the same patients presenting the active disease and after anti-malarial therapy and parasitological cure or to HD. (Figure 1). The levels of TNF-α in the sera did not differ significantly. The levels of IL-10 were also higher before treatment than after parasitological cure. No significant differences in the level of circulating cytokines were found between HD (IL-8 median: 9.8; interquartile (IQR): 1.8- 21; IL-1β median: 0.2 IQR: 0.13–2.8; IL-6 median: 0.14; IQR:0–5; IL-10 median: 0–7.1; IQR: 0–7 and TNF-α median: 5.4; IQR 0–21) and malaria patients who had completed the chemotherapy.
The cytokines IL-8 (CXCL8), IL-1β, IL-6, IL-10, TNF-α were measured in the plasma of P. vivax-infected subjects (n = 26), before (closed circles) and 30–45 days after treatment (open circles). Dotted lines represent medians of given measurements from healthy donors (HD; n = 13). Levels of cytokines were measured employing the Cytometric Bead Array (CBA). Significant differences are indicated with p-values using Wilcoxon signed rank test when the data failed the normality test.
Monocytes and neutrophils activation during P. vivax infection
The expression of various activation markers was analyzed on monocytes (Figure 2A) and neutrophils (Figure 2B). Monocytes were gated taking in consideration the expression of CD14, as shown in the representative density plot (Figure 2A). Lower expression of HLA-DR (median fluorescence intensity-MFI) was observed on monocytes from 9 out of 12 P. vivax-infected patients, when compared to the same patients after anti-malarial treatment. To assess the neutrophil population, CD66b+CD16+ cells were gated. As CD66b is GPI-anchores highly glycoslated protein, widely expressed on human neutrophils and to regulate neutrophil acitvation. Lower levels of CD62L (8 out of 12 subjects) and CD88 (all patients) were found on neutrophils from malaria patients before treatment (Figure 2B). The lower expression of HLA-DR as well as CD62L is a cell adhesion molecule, and CD88 is a G protein-coupled receptor for C5a, have been previously described in activated monocytes and neutrophils, respectively [14], [19]. The levels of these activation markers return to normal levels (comparable to the median of HD (monocytes: HLA-DR (median: 278; IQR: 143–323); neutrophils: CD88 (median: 27; IQR: 17–32) and CD62L (median: 601; IQR: 439–666); in cells from P. vivax-patients submitted to anti-malarial therapy.
Mean fluorescence intensity (MFI) of HLA-DR, TLR2 and TLR4 was evaluated on monocytes (A) and CD62L, CD88, TLR2 and TLR4 on neutrophils (B) in whole blood from P. vivax-infected subjects (n = 12), before (closed circles) and 30–45 days after treatment (open circles). Representative density plots showing the gate strategy for monocytes CD14+ (A) and neutrophils CD66b+CD16+ (B) are shown. Significant differences are indicated with p-values using Wilcoxon signed rank test when the data failed the normality test.
Monocytes are a major source of cytokines during P. vivax malaria
The expression of TLR2 was lower on both monocytes (8 out of 11 subjects) and neutrophils (10 out of 11 subjects) from malaria patients, whereas expression of TLR4 was only affected on neutrophils (8 out of 12) from infected individuals (Figures 2A and 2B). After parasitological cure, the surface expression of TLR returned to levels comparable to cells from HD (monocytes: TLR2 (median: 256; IQR: 241–276) and TLR4 (median: 11; IQR: 9–17; neutrophils: TLR2 (median 46; IQR: 36–62) and TLR4 (median: 3.5; IQR: 2.9–6.4)). Two millions per ml (2×105/well) of highly purified monocytes or neutrophils were cultured with or without TLR agonists and cytokines measured in the tissue culture supernatants (Figure 3A and B). Cell preparations reached over 98% of purity, as shown in the representative dot plots presented in Figure 3. Except for IL-8, neutrophils were over all a poor source of cytokines. No detectable levels of IL-1β, IL-6, IL-10 and TNF-α were found in culture supernatants of neutrophils stimulated or not with LPS or Pam (Figure 3B), even when neutrophils were used at a concentration of 1×107/ml (1×106/well). On the other hand, upon stimulation with LPS or Pam, high levels of IL-1β, IL-6, and TNF-α were produced by monocytes from malaria patients. (Figure 3A). Interestingly, the levels of LPS-induced IL-10 were lower in culture supernatants of monocytes derived from patients with ongoing P. vivax infection. After parasitological cure the cytokine production by monocytes returned to levels comparable to those produced by monocytes from HD (median: 1692; IQR: 1200–5265). IL-8 (CXCL8) was the only cytokine produced in high levels by neutrophils. Despite the down-modulation of TLR2 and TLR4, stimulation with TLR agonists increased the levels of IL-8 produced by neutrophils (Figure 3B) from malaria patients before (medium versus LPS, p = 0.0010 or Pam, p = 0.0024) and after treatment (medium vs LPS, p = 0.0002; or Pam, p = 0.0017). IL-8 was also produced by monocytes and the levels of cytokine did not differ between patients, before and after treatment (Figure 3A).
Purified monocytes (A) or neutrophils (B) from P. vivax-infected subjects before (closed circles; n = 13) and 30–45 days after treatment (open circles; n = 13) were cultured for 48 hours in the absence or presence of LPS or Pam. Levels of IL-1β, IL-6, IL-10, TNF-α, and IL-8 (CXCL8) were measured in supernatant of monocyte (A) and neutrophil (B) cultures. Levels of cytokines were measured employing the Cytometric Bead Array (CBA). Significant differences are indicated p-values using paired t test or Wilcoxon signed rank test when a normality test failed.
Enhanced oxidative and phagocytic activity by neutrophils from malaria patients
The O2− production by neutrophils was increased during acute malaria (Figure 4). Higher percentage of NBT positive cells was observed in the blood of P. vivax-infected patients (Figure 4, left panel). The function of neutrophils was further investigated by assessing their phagocytic activity, which was also higher in cells from P. vivax-infected individuals (Figure 4, right panel).
Neutrophils were isolated from P. vivax-infected patients (closed circles; n = 15) or healthy donors (open circles; n = 15), and the frequencies of neutrophils reducing NBT (left panel) as well as cell containing zymosan (right panel) were quantified. Significant differences are indicated with p-values using unpaired t test or Mann-Whitney test when a normality test failed.
Impaired chemotaxis by neutrophils from malaria patients
The ability of purified neutrophils to migrate towards CXCR1, CXCR2 and CCR2 ligands (IL-8 and CCL2) was evaluated by using a modified Boyden chamber assay [20]. Neutrophils from malaria patients showed impaired chemotaxis to IL-8, when compared to HD (median: 31; IQR: 28–39) (Figure 5A). The poor migration of neutrophils from malaria patients was associated with decreased expression of the chemokine receptor CXCR2, as indicated by qPCR (Figure 5C) and FACS (Figure 5B). Chemotaxis to CCL2 and expression of CCR2 by neutrophils were minimal and not altered in malaria patients (Figures 5A and 5B).
Neutrophils were isolated from P. vivax-infected patients (closed circles; n = 15) or healthy donors (open circles; n = 15), and chemotaxis towards IL-8 (CXCL8) and CCL2 was assessed (A). MFI of CXCR1, CXCR2 and CCR2 on neutrophils were evaluated by flow cytometry and representative histograms of CXCR2 expression are shown (B). CXCR2 message was measured by qPCR (C). Significant differences are indicated with p-values using unpaired t test or Mann-Whitney test when a normality test failed.
Enhanced expression of GRK2 in neutrophils from malaria patients
GRK2 has been described to down-modulate expression of chemokine receptors. Importantly, continuous and excessive activation of neutrophils results in an increased expression of GRKs, which phosphorylate G protein–coupled receptors (GPCR) leading to receptor desensitization [21]. GRK2 expression was measured by immunofluorescence in neutrophils purified from blood of P. vivax-infected patients and HD (median: 12.6; IQR: 4.7–23). Compared with HD, a significant increase in GRK2 expression was observed in neutrophils from P. vivax-infected patients (Figure 6A and 6B).
Neutrophils isolated from P. vivax-infected patients (closed circles; n = 11) or healthy donors (closed circles; n = 12) were stained for GRK2 and mean fluorescence intensity (MFI) of GRK2 was quantified (A). Representative fluorescence microscopy illustrating GRK2 expression in neutrophils from a healthy donor and a P. vivax-infected patient (B). Significant difference is indicated with p-values using unpaired t test.
The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators [1], [22]. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. Neutrophils are the most abundant leukocyte population in the peripheral blood and thus being in direct contact with red blood cells, the main cells population that is parasitized by Plasmodium species. Surprisingly, very few studies have addressed the role of the polymorphonuclear cells (PMNs) during malaria. In this study, we investigated the activation and function of neutrophils during acute episodes of P. vivax malaria. Neutrophils were shown to be highly activated presenting enhanced phagocytic activity as well as superoxide production. In contrast, neutrophils were found to be poor sources of cytokines during malaria. It is noteworthy that we described for the first time an impaired chemotaxis by circulatory neutrophils from malaria patients. Importantly, different studies have described an association of severe malaria and bacterial infections [23], [24]. Thus, our results suggest that the failure of neutrophils to migrate to the sites of infection may represent an important mechanism that leads to enhanced susceptibility of malaria patients to secondary bacterial infections.
We first noticed that both circulatory neutrophils (decreased expression of CD62L and CD88) and monocytes (decreased expression of HLA-DR) from malaria patients were highly activated. We then asked, what would be the primary source of pro-inflammatory cytokines during P. vivax-malaria? We found that in the blood, monocytes are an important source of cytokine compared with neutrophils. Despite the fact that neutrophils are an important source of cytokines [25], we found that except for IL-8 (CXCL8), the neutrophils from malaria patients produced none or very small amounts of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-αin response to TLR agonists. Importantly, PBMCs from individuals experimentally or naturally infected with P. falciparum are hyperresponsive and produce high amounts of pro-inflammatory cytokines once activated with TLR agonists [6], [7]. Here, we observed that highly purified monocytes (but not neutrophils) derived from P. vivax malaria patients were primed and produce high levels of IL-1β, IL-6 and TNF-α upon TLR stimulation. In contrast, the monocytes from malaria patients produced low levels of IL-10, even when activated with TLR agonists. Thus, we favor the hypothesis that during malaria monocytes differentiate into an inflammatory stage producing high levels of pro-inflammatory cytokines and low levels of IL-10.
On the other hand, circulatory neutrophils from malaria-patients displayed enhanced phagocytic activity and constitutively released high levels of superoxide. The process of neutrophil activation could involve phagocytosis of opsonized parasites, which would in turn trigger the antibody dependent respiratory burst [26]. Alternatively, phagocytosis of the malaria pigment (hemozoin) may also activate neutrophils [27]. The enhanced effector function of neutrophils may account for a more efficient up-take and destruction of free parasites and infected erythrocytes [26]. On the other hand, activated neutrophils have been shown to cause damage of endothelial cells, in a process that is mediated by sera of malaria patients [28]. Therefore, enhanced effector functions in neutrophils could be involved in both host resistance and pathogenesis of P. vivax malaria.
Unexpectedly, we found an altered migration towards IL-8 gradient, which was associated with a decreased expression of CXCR2 on neutrophils from P. vivax-infected patients. Functional studies showed that upon phagocytosis of bacteria by neutrophils there is reduction in expression of CXCR1 and CXCR2 [29]. Down-regulation of CXCR2 in severe sepsis also results in failure of neutrophil migration that is associated with enhanced susceptibility to bacterial infection [30]. Furthermore, expression of CD88 (C5a receptor) and CD62L (L-selectin) is decreased on the surface of neutrophils from P. vivax-malaria patients. CD88 is a G protein-coupled receptor involved in recruitment [19], [31], whereas CD62L is a key molecule that mediates cytoadherence of leukocytes [32]. Thus, altogether, our data strongly suggest that systemic activation neutrophils, leads to failure of extravasation and chemotaxis from blood to the tissues. Since IL-8 mediates chemotaxis and stimulate neutrophils to release specific granules and proteases to fight microbial infections [33], [34], the impairment of neutrophil migration to the site of infection would prevent front line cells to promote an inflammation to effectively kill infectious pathogens allowing secondary infections.
Production of IL-8 has been assessed in several cell type upon stimulation, several medical conditions and even constitutively [35], [36], [37]. The observed high levels of circulatory IL-8 may mediate desensitization and/or down-regulation of CXCR2 in acutely infected malaria patients. In addition, TNF-α [38], nitric oxide [30], heme-oxygenase products [39] and TLR ligands [20], cause the heterologous desensitization of CXCR2 via GRK2 induction. As previous described in vivax malaria [40], [41], we did not find a high level of TNFα in serum, the monocyte stimulated with TLR agonists produced high amounts of TNF-α that may contribute to CXCR2 desensitization. In addition, Plasmodium can be recognized by TLR2, TLR4 and TLR9 [10], [42] and induce down-regulation of CXCR2 via GRK2. Importantly, CD88 is also desensitized via GRK2 [43]. Thus, decreased expression of CXCR2 and CD88 on neutrophils from malaria patients may be a consequence of an enhanced expression of GRK2.
GRKs constitute a group of serine/threonine protein kinases that are key modulators of protein-coupled receptor signaling (GPCR) [44]. A major mechanism for desensitization of activated GPCR is their phosphorylation by GRKs [45]. Of note both CD88 and CXCR2 are GPCRs. Deficient expression of GRK and regulation of chemokine receptors in GRK2+/− mice results in enhanced migration of lymphocytes and chemotaxis toward CCL4, the CCR5 ligand [46]. It was also described that transcription of GRK2 and GRK5 is upregulated upon LPS-mediated activation, leading to reduced expression of chemokine receptor and neutrophils chemotaxis [47]. GRK2 and GRK5 expression are enhanced in sepsis patients [48] and in rodent models of severe sepsis [20], which are associated with impaired migration of neutrophils and enhanced susceptibility to secondary microbial infection.
For many years, P. vivax malaria was considered a benign and self-limited disease, especially when compared to P. falciparum infection [49]. However, recent studies highlighted the association of P. vivax malaria with life-threatening manifestations, such as respiratory distress, severe thrombocytopenia and anemia, as well as neurological manifestations [1], [22], [50], [51], [52], [53], [54]. A main hypothesis of our research group is that secondary infections, in malaria primed individuals, is a main cause of severe disease. In this regard, pro-inflammatory priming during malaria would result in dramatic decrease in the threshold to initiate a septic shock [7], due to an over-reaction to secondary infections, particularly in the case of bacteria that have extremely potent TLR agonists.
Importantly, areas of the world with the highest incidence and prevalence of malaria also have a high incidence of bacterial infections, including Salmonella, Pneumococcus and Meningococcus [23], [55]. Furthermore, a recent study highlights that severe malaria as indicated by respiratory distress, anemia and mortality, is 8.5 times more elevated in children with both malaria and bacteremia as compared to infection with P. falciparum alone [24]. Thus, co-infection with bacteria is not only common, but as we might predict, it is an important factor influencing outcome of disease and development of severe disease [24], [56]. Here, we demonstrate for the first time that circulatory neutrophils from malaria patients display a decreased expression of chemokine receptors and adhesion molecules, which culminates in impaired chemotaxis. Hence, our results suggest that a failure of these PMNs to migrate to peripheral tissues is an important mechanism leading to enhance susceptibility to bacterial infection during malaria.
We are grateful to Dr. Marco Cassatella for all advice and for critically reading this manuscript, to Dr. Luiz Hildebrando Pereira da Silva for supporting the development of this work in the endemic area of Porto Velho and also to the nurses for excellent technical assistance, and to the Program for Technological Development in Tools for Health–PDTIS-FIOCRUZ for use of its facilities.
Author Contributions
Conceived and designed the experiments: FMSL LRVA RTG. Performed the experiments: FMSL LRVA SCT BCR PACC HDG. Analyzed the data: FMSL LRVA SCT. Contributed reagents/materials/analysis tools: FMSL LRVA RTG DTG FQC MST DBP. Wrote the paper: FMSL LRVA RTG. Assisted with patient care and case identification: MST DBP.
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- 10. Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, et al. (2005) Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 280: 8606–8616.
- 11. Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, et al. (2007) Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci U S A 104: 1919–1924.
- 12. Franklin BS, Ishizaka ST, Lamphier M, Gusovsky F, Hansen H, et al. (2011) Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria. Proc Natl Acad Sci U S A 108: 3689–3694.
- 13. Sharma S, DeOliveira RB, Kalantari P, Parroche P, Goutagny N, et al. (2011) Innate immune recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum genome. Immunity 35: 194–207.
- 14. Schwarzer E, Alessio M, Ulliers D, Arese P (1998) Phagocytosis of the malarial pigment, hemozoin, impairs expression of major histocompatibility complex class II antigen, CD54, and CD11c in human monocytes. Infect Immun 66: 1601–1606.
- 15. Skorokhod OA, Alessio M, Mordmuller B, Arese P, Schwarzer E (2004) Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: a peroxisome proliferator-activated receptor-gamma-mediated effect. J Immunol 173: 4066–4074.
- 16. Jaramillo M, Plante I, Ouellet N, Vandal K, Tessier PA, et al. (2004) Hemozoin-inducible proinflammatory events in vivo: potential role in malaria infection. J Immunol 172: 3101–3110.
- 17. Mohammed AO, Elghazali G, Mohammed HB, Elbashir MI, Xu S, et al. (2003) Human neutrophil lipocalin: a specific marker for neutrophil activation in severe Plasmodium falciparum malaria. Acta Trop 87: 279–285.
- 18. Kimura M, Kaneko O, Liu Q, Zhou M, Kawamoto F, et al. (1997) Identification of the four species of human malaria parasites by nested PCR that targets variant sequences in the small subunit rRNA gene. Parasitol Int 46: 91–95.
- 19. Fortunati E, Kazemier KM, Grutters JC, Koenderman L, Van den Bosch JM (2009) Human neutrophils switch to an activated phenotype after homing to the lung irrespective of inflammatory disease. Clin Exp Immunol 155: 559–566.
- 20. Alves-Filho JC, Freitas A, Souto FO, Spiller F, Paula-Neto H, et al. (2009) Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis. Proc Natl Acad Sci U S A 106: 4018–4023.
- 21. Gainetdinov RR, Bohn LM, Walker JK, Laporte SA, Macrae AD, et al. (1999) Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice. Neuron 24: 1029–1036.
- 22. Karunaweera ND, Wijesekera SK, Wanasekera D, Mendis KN, Carter R (2003) The paroxysm of Plasmodium vivax malaria. Trends Parasitol 19: 188–193.
- 23. Bronzan RN, Taylor TE, Mwenechanya J, Tembo M, Kayira K, et al. (2007) Bacteremia in Malawian children with severe malaria: prevalence, etiology, HIV coinfection, and outcome. J Infect Dis 195: 895–904.
- 24. Were T, Davenport GC, Hittner JB, Ouma C, Vulule JM, et al. (2011) Bacteremia in Kenyan children presenting with malaria. J Clin Microbiol 49: 671–676.
- 25. Cassatella MA (1999) Neutrophil-derived proteins: selling cytokines by the pound. Adv Immunol 73: 369–509.
- 26. Joos C, Marrama L, Polson HE, Corre S, Diatta AM, et al. (2010) Clinical protection from falciparum malaria correlates with neutrophil respiratory bursts induced by merozoites opsonized with human serum antibodies. PLoS One 5: e9871.
- 27. Huy NT, Trang DT, Kariu T, Sasai M, Saida K, et al. (2006) Leukocyte activation by malarial pigment. Parasitol Int 55: 75–81.
- 28. Hemmer CJ, Vogt A, Unverricht M, Krause R, Lademann M, et al. (2008) Malaria and bacterial sepsis: similar mechanisms of endothelial apoptosis and its prevention in vitro. Crit Care Med 36: 2562–2568.
- 29. Doroshenko T, Chaly Y, Savitskiy V, Maslakova O, Portyanko A, et al. (2002) Phagocytosing neutrophils down-regulate the expression of chemokine receptors CXCR1 and CXCR2. Blood 100: 2668–2671.
- 30. Rios-Santos F, Alves-Filho JC, Souto FO, Spiller F, Freitas A, et al. (2007) Down-regulation of CXCR2 on neutrophils in severe sepsis is mediated by inducible nitric oxide synthase-derived nitric oxide. Am J Respir Crit Care Med 175: 490–497.
- 31. Azeredo EL, Neves-Souza PC, Alvarenga AR, Reis SR, Torrentes-Carvalho A, et al. (2010) Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever. Immunology 130: 202–216.
- 32. Ley K (2002) Integration of inflammatory signals by rolling neutrophils. Immunol Rev 186: 8–18.
- 33. Larsen CG, Anderson AO, Appella E, Oppenheim JJ, Matsushima K (1989) The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes. Science 243: 1464–1466.
- 34. Baggiolini M, Dewald B, Moser B (1994) Interleukin-8 and related chemotactic cytokines–CXC and CC chemokines. Adv Immunol 55: 97–179.
- 35. Srivastava MD, Srivastava R, Srivastava BI (1993) Constitutive production of interleukin-8 (IL-8) by normal and malignant human B-cells and other cell types. Leuk Res 17: 1063–1069.
- 36. Wurm S, Neumeier M, Weigert J, Wanninger J, Gerl M, et al. (2008) Insulin induces monocytic CXCL8 secretion by the mitogenic signalling pathway. Cytokine 44: 185–190.
- 37. Wong CK, Wong PT, Tam LS, Li EK, Chen DP, et al. (2010) Activation profile of Toll-like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus. Clin Exp Immunol 159: 11–22.
- 38. Secher T, Vasseur V, Poisson DM, Mitchell JA, Cunha FQ, et al. (2009) Crucial role of TNF receptors 1 and 2 in the control of polymicrobial sepsis. J Immunol 182: 7855–7864.
- 39. Freitas A, Alves-Filho JC, Trevelin SC, Spiller F, Suavinha MM, et al. (2011) Divergent role of heme oxygenase inhibition in the pathogenesis of sepsis. Shock 35: 550–559.
- 40. Andrade BB, Reis-Filho A, Souza-Neto SM, Clarencio J, Camargo LM, et al. (2010) Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance. Malar J 9: 13.
- 41. Goncalves RM, Salmazi KC, Santos BA, Bastos MS, Rocha SC, et al. (2010) CD4+ CD25+ Foxp3+ regulatory T cells, dendritic cells, and circulating cytokines in uncomplicated malaria: do different parasite species elicit similar host responses? Infect Immun 78: 4763–4772.
- 42. Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, et al. (2005) Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J Exp Med 201: 19–25.
- 43. Langkabel P, Zwirner J, Oppermann M (1999) Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by "G protein-coupled receptor kinases. Eur J Immunol 29: 3035–3046.
- 44. Penela P, Ribas C, Mayor F Jr (2003) Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases. Cell Signal 15: 973–981.
- 45. Krupnick JG, Benovic JL (1998) The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu Rev Pharmacol Toxicol 38: 289–319.
- 46. Vroon A, Heijnen CJ, Lombardi MS, Cobelens PM, Mayor F Jr, et al. (2004) Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4. J Leukoc Biol 75: 901–909.
- 47. Fan J, Malik AB (2003) Toll-like receptor-4 (TLR4) signaling augments chemokine-induced neutrophil migration by modulating cell surface expression of chemokine receptors. Nat Med 9: 315–321.
- 48. Arraes SM, Freitas MS, da Silva SV, de Paula Neto HA, Alves-Filho JC, et al. (2006) Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation. Blood 108: 2906–2913.
- 49. Singh H, Parakh A, Basu S, Rath B (2011) Plasmodium vivax malaria: is it actually benign? J Infect Public Health 4: 91–95.
- 50. Anstey NM, Handojo T, Pain MC, Kenangalem E, Tjitra E, et al. (2007) Lung injury in vivax malaria: pathophysiological evidence for pulmonary vascular sequestration and posttreatment alveolar-capillary inflammation. J Infect Dis 195: 589–596.
- 51. Obaldia N 3rd (2007) Clinico-pathological observations on the pathogenesis of severe thrombocytopenia and anemia induced by Plasmodium vivax infections during antimalarial drug efficacy trials in Aotus monkeys. Am J Trop Med Hyg 77: 3–13.
- 52. Anstey NM, Russell B, Yeo TW, Price RN (2009) The pathophysiology of vivax malaria. Trends Parasitol 25: 220–227.
- 53. Alexandre MA, Ferreira CO, Siqueira AM, Magalhaes BL, Mourao MP, et al. (2010) Severe Plasmodium vivax malaria, Brazilian Amazon. Emerg Infect Dis 16: 1611–1614.
- 54. Bassat Q, Alonso PL (2011) Defying malaria: Fathoming severe Plasmodium vivax disease. Nat Med 17: 48–49.
- 55. Berkley JA, Maitland K, Mwangi I, Ngetsa C, Mwarumba S, et al. (2005) Use of clinical syndromes to target antibiotic prescribing in seriously ill children in malaria endemic area: observational study. Bmj 330: 995.
- 56. Berkley JA, Bejon P, Mwangi T, Gwer S, Maitland K, et al. (2009) HIV infection, malnutrition, and invasive bacterial infection among children with severe malaria. Clin Infect Dis 49: 336–343.


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Government Websites
CFPB developed the eRegulations site as an alternative to paper editions of regulations, guidebooks, multiple websites and paid subscription services.
A new website from the Consumer Financial Protection Bureau lays out the agency's written regulations in a single, navigable interface. The site is aimed at compliance officers, attorneys and CFPB employees. But the creation of the one-stop, open-source platform could have implications for other agencies looking to make their information more accessible.
For interested parties, eRegulations offers an alternative to clunky paper editions of regulations, guidebooks, multiple websites and paid subscription services. The agency maintains rules on consumer-facing financial products and services, and regulations on topics ranging from required disclosures for automatic teller machine transactions to rules for overdraft services at banks.
Especially for smaller or rural institutions, keeping up with the law can be a time consuming and expensive proposition.
"A compliance officer at a small community bank shouldn't have to sign up for a service to find out what the regulations are," said Matt Burton, acting CIO in the Technology Innovation office at CFPB.
The process started about a year ago, when the regulations office asked the technology team to work on a product that would streamline the experience of reading regulations. But the real story of eRegulations could be the opportunities opened up to other agencies looking to publish their rules online. Underneath the site, said Burton, "is an [application programming interface] that will ingest a body of regulations and spit them out as structured data."
There's a built in structure to a regulatory code, with chapters, paragraphs, and subparagraphs. The CFPB's tool parses the text of regulations a machine-readable collection of data that can be displayed in web form with each subparagraph having its own unique URL for handy reference, and with individual regulations strung together in order so a user can page through. There are links to supporting material, including interpretations of rules and supporting statutes.
"Our hope is that other agencies will see this and use it as their own regulations platform," Burton said. Because the eRegulations is open source, independent developers could draw on it as the basis for tools displaying government information. No agency has expressed an interest yet, but the site and the underlying API were only released this week.
About the Author
Adam Mazmanian is a staff writer covering Congress, the FCC and other key agencies. Connect with him on Twitter: @thisismaz.
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President Obama's proposal to boost government coordination with the private sector got a warm welcome in the House Homeland Security Committee.
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Meet the women and men who are doing great things in federal IT.
The law's certification and approval provisions empower CIOs to end outdated software development projects, says Agilex's Roger Baker.
The new chairman of the House Oversight and Government Reform Committee extends an olive branch to the minority, but keeps subpoena power for himself.
FCW investigated efforts by the departments of Defense and Veterans Affairs to improve a joint data repository on military and veteran suicides. Something as impersonal and mundane as incomplete datasets could be exacerbating a national tragedy.
Despite delays, the program is at a critical point for determining the ultimate impact of cloud technology in the government space.
In an interview with FCW, the Department of Veterans Affairs' chief technology officer talks about overhauling the digital experience for VA customers.
The National Information Exchange Model's usefulness extends far beyond its origins in justice and law enforcement.
I just forwarded this to my program director for his consideration.

subset: commoncrawl


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Need help with Part B unable to get teh answer.
Image text transcribed for accessibility: Consider a Vex multiplier with R1 = R2 = 1.2kOhm, utilizing a transistor that has Vex = 0.6 V at IC = 1 mA, And a very high beta. (a) Find the value of the current I that should be supplied to the multiplier to obtain a terminal voltage of 1.2 V. (b) Find the value of I that will result in the terminal voltage changing (from the 1.2V value) by +50 mV, +100 mV, +200 mV, -50mV, -100 mV, -200 mV. 1.5 mA; (b) 3.24 mA, 7.93 mA, 55.18 mA, 0.85 mA, 0.59 mA, 0.43 mA


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Serving up all the latest food news, cool foodie products, gossip, and good deals that are fit to print!
Ron Burgundy Blended Scotch Whiskey — It's Real, and You Can Buy It
Everyone knows Ron Burgundy is a big fan of his "scotchy scotch scotch," and with the return of Anchorman to the big screens this month comes a Ron Burgundy-inspired whiskey. According to Foodbeast, the product is aptly named Ron Burgundy Blended Scotch Whiskey. The bottle's label calls it a "Great Odin's Raven Special Reserve." The blended whiskey features spirits from Speyside, Highlands, and Islay in Scotland. Ron Burgundy devotees will be able to pick up a bottle sometime this month, just in time for the release of Anchorman 2: The Legend Continues.
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ELK Lighting 86052 3-Light Huarco Small Chandelier
Product Description
Available while supplies last.
This ELK Lighting product has a seashell finish. Features amber glass. For use with three 40-watt halogen bulbs.
The true source of the Amazon was discovered in 1953 to be a small stream, the Huarco, in the Andes Mountains of Peru. The thick canopy of foliage that surrounds this meandering waterway was the inspiration for this small chandelier.
- The refreshing green leaves invoke a feeling of a tropical rain forest.
- Complimenting the month-blown glass, the base of each blossom is adorned with chestnut hues clasping clear crystal droplets.
Finish: | Seashell | |
Glass: | Amber | |
Light Bulb: | (3)40w T4 G9 120v Halogen | |
Voltage: | 120 volt | |
Weight: | 6 Pounds | |
Installation: | Dry Locations [Explain] | |
Listing: | , Quick Ship | |
Specialty Chandelier Type Chandeliers come in a wide variety of shapes and styles. More common types include Candle-Style, Crystal, Williamsburg, and Wrought Iron among others. | Specialty Chandelier Type | Mini Chandelier |
Bulb is included free
Bulbs are not eligible for replacement
Bulbs are not eligible for replacement
Dimensions & Weights
Wire Length: | 6 feet |
Diameter: | 23 inches |
Shade Diameter: | 4 inches |
Body Height: | 16 inches |
Canopy Diameter: | 5 inches |
Chain/Cord: | 36 inches |
Height: | 4 feet 4 inches |
Shade Height: | 4 inches |
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5 out of 5
Write a ReviewBreakfast Room close to perfection
By Melanie T. from mobile, AL on Sep 04, 2008
The light fixture is as lovely in person as it was on the page, with the exception of the "goldness" of the crystal grape holders. It appeared a little more purple in the picture, but it will look wonderful in my home!!
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By joel c. from wellington, FL on Mar 24, 2011
Bargain Hunter
The product that I bought the elk chandelier is awesome I had no trouble installing it was very easy to put up It was the correct size shape color everything I thought it would be It goes perfect with my island theme I have going on in my house I would recommend as product anyone
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Wonderful product
By Ann D. from Homosassa, FL on Jan 27, 2010
I love the look of the fixture....everyone that has seen has remarked how beautiful and well made and different. Kudos
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By Marilyn R. from York, PA on Oct 12, 2009
A lovely chandelier, just the right size for my dining room. Gives off lots of light although it is a little too harsh. Wish it was more in the yellow range and not so white. Have had many compliments on it.
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Group # | 289896 |
Category | Chandeliers - 89 |
Style | Country Cottage - 287 |
Sales Rank | 291 |
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Chef Norman Van Aken and Chef Ron DeBonis are showing YOU how to prepare some of the dishes that will be featured in Busch Gardens' inaugural Food & Wine Festival debuting on March 7. Take the next 90 seconds and learn how to make this Goat Cheese Potato Tart with Pistachio Oil, which will be featured in the Chef's Plantation food cabin.
Busch Gardens' oldest chimpanzee, Patsy, will be turning 53 years old on March 1 and the animal care team is celebrating Mardi Gras-style! The celebration will begin at 1 p.m. at Myombe Reserve with presents, "cake" and other enrichment activities, including masks and fleur de les stuffed with treats. Plus, a birthday party isn't complete without a birthday banner!
Busch Gardens Tampa has welcomed two new adorable additions – three-month-old cheetah cubs. The Busch Gardens animal care team is providing 24-hour-care as the cubs continue to get stronger and explore their new home.
The cubs were born on Nov. 22, 2014 and weigh approximately 12 pounds. Their names, which were given by the Busch Gardens animal care team, are Tendai, meaning "thankful," and Thabo, meaning "joy."
Summer will be here in no time and although work continues for parents, the fun is just beginning for kids! Start planning ahead and book an amazingly wild adventure for your child this summer with Busch Gardens resident camps.
It seems like just yesterday Kasi was an adorable ball of fluff scampering about Cheetah Run at Busch Gardens. My how time does fly! Join us in celebrating Kasi's offical adulthood during his 4th birthday celebration on February 19.
Busch Gardens� Tampa has welcomed another endangered western lowland gorilla. The one-week-old female gorilla was born on Feb. 6 at 8:30 p.m. and is being cared for by 27-year-old mother Mary. This birth brings the gorilla troop to seven.
Get a first look at the baby here:
Maybe some up-close animal encounters, activities and curriculum you can take back to your classroom, a sunrise safari, and time to connect with other fun-loving teachers over dinner and drinks?
By Matt Turner


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2012 1/4 oz Proof Gold Heroes Literature d'Artagnan (Musketeer)
Product Details
Mintage of just 1,000 coins!
This is the second year of the series which pays tribute to characters of French literature. This year the adventurers are in the spotlight. The second coin this year depicts d'Artagnan.
d'Artagnan was one of the musketeers under the reign of King Louis XIII. The Three Musketeers is a novel by Alexandre Dumas which recounts the adventures of d'Artagnan.
The obverse of this Gold coin presents d'Artagnan in all the splendor of his period costume. In the background, the three musketeers declare their famous motto "Un pour tous, tous pour un" ("all for one, one for all"). The reverse has a portrait of Alexandre Dumas. Included is the quote from the play: "Le jeune Mousquetaire etait en excellente disposition pour trepasser heroiquement". Each of these beautiful Gold coins comes in a box and with a certificate of authenticity.
Gold -- why buy Gold from APMEX?
Gold is one of the world's favorite bullion investments and is a popular way to diversify one's wealth. Call our Account Managers toll free at (800) 375-9006 or click on the "Live Chat" button. APMEX is where to buy Precious Metals! Also, you can come to us when it is time to sell Gold, Silver, Platinum, and Palladium.Specifications
Product ID: | 73386 |
Year: | 2012 |
Grade: | Proof |
Grade Service: | None |
Denomination: | €50 |
Mint Mark: | None |
Metal Content: | 0.25 troy oz |
Purity: | .920 |
Manufacturer: | Monnaie de Paris |
Diameter: | 22 mm |


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WASHINGTON – The Office of the Comptroller of the Currency today announced that Deputy Comptroller for Credit and Market Risk Darrin Benhart will co-chair the Task Force on Prudential Treatment of Assets of the Basel Committee on Bank Supervision.
The task force will study the use by banks and supervisors of such terms as "non-performing loan," "loss," "write-off," and "forbearance," and possibly develop common international definitions as appropriate. During asset classification, the recognition of loss and write-off depends on identifying those assets and portions of assets that should be considered uncollectable or of insignificant value. A better understanding of these factors across jurisdictions would benefit risk disclosures and financial reporting. Mr. Benhart's co-chair on the task force is Mr. Piers Haben, Director of the Oversight Department of the European Banking Authority.
"I am proud that international organizations turn to seasoned supervisors of the Office of the Comptroller of the Currency for their expertise and leadership," said Comptroller of the Currency Thomas J. Curry. "Darrin's deep experience and knowledge will serve the agency and the Basel Task Force well in this important role."
Search Office of Thrift Supervision (OTS) Archive


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Item |
Set: 2013-14 ITG Used Autographed Memorabilia |
Player: Smith, Billy |
Sport: Hockey |
Brand: ITG Used |
Manufacturer: In the Game |
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Hotel Holiday Inn Express Niles(2 stars)
1000 Moore St View on mapUnfortunately there are no hotel reviews available. Be the first to review Hotel Holiday Inn Express Niles and help other guests.
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Travelers looking for excellent quality at a great price "Stay Smart" at our brand new Holiday Inn Express® Niles, MI hotel. Located just 8 miles north of The University of Notre Dame and South Bend area. Our hotel offers the best in service and amenities for business and leisure travelers. Experience a carefree stay at our Niles, MI hotel which is the only hotel with an indoor pool, fitness center and meeting space. Take advantage of the pool after a long day of meetings. We have everything a business traveler needs for a productive trip. Keep in touch with the office using complimentary Wi-Fi access or at a computer in the Business Center. You can use the 1100-sq-ft meeting room to host an event or seminar for up to 75 attendees. Our hotel's location in the Niles - South Bend area is ideal for work and pleasure. Our 100% non-smoking facilities are filled with amenities to help you unwind completely. Test the waters of our heated, indoor pool or just sit back in the privacy of your own room and soak in your private whirlpool tub. Our Fitness Center offers you the convenience of your daily workouts, and each morning you can enjoy a hot, tasty deluxe breakfast on the house. Let us show you the unparalleled hospitality of Niles, MI.Booking and cancellation policies vary by rate type. Please check all applicable rules prior to booking to avoid possible charges. American Disabilities Act. If your accessibility needs are not met, please contact the Hotel Manager on Duty. Should you require additional information regarding accessible facilities for guests with disabilities, please call 1-800-465-4329 (U.S. and Canada) or contact the hotel directly. For the hearing impaired, TDD service is available at 1-800-238-5544 within the U.S. and CanadaParking
* Neither HolidayCheck nor the provider assumes liability for the accuracy and completeness of the content displayed above. Please take note of the hotel description of the respective operator and bear in mind that the services included in an offer may differ from the catalog description presented here. We are obliged to offer solely those services available at the time of booking as shown on the booking form. © 2013 Giata
Hotel amenities
- Meeting/Banquet Facilities
- Non-smoking room
- Shops in the hotel
- Business centre
- Hotel safe
- Air conditioning
- Free parking
- Car park
Room amenities
- Writing bureau
- Additional childrens' bed
- Safe
- Coffee and tea
- Bathroom
- Wake up call/Alarm clock
- Restaurant
A choice of sports
- Fitness centre
- Outdoor pool
Spa offers
- Whirlpool
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- Babysitting/Child services
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- All public and private areas are no smoking zones
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Radian Agrees To Commute $827 Million Financial Guaranty Reinsurance Portfolio To FGIC
Radian Group Inc. today announced that on November 09, 2012, its financial guaranty insurance subsidiary, Radian Asset Assurance Inc., entered into an agreement (the "Agreement") with Financial Guaranty Insurance Company ("FGIC") which, if consummated, would commute the remaining $827 million of outstanding par reinsured by Radian Asset from FGIC (the "Commutation"). This portfolio represents 13 percent of Radian Asset's total reinsurance exposure as of September 30, 2012, and includes $196 million of Radian Asset's $225 million in net par outstanding as of September 30, 2012, related to Jefferson County, Alabama sewer warrants.
"Entering into this agreement is another example of our efforts to actively reduce our financial guaranty exposure, which is an important component of our capital management strategy," stated Chief Executive Officer S.A. Ibrahim.
A rehabilitation proceeding for FGIC pursuant to Article 74 of the New York Insurance Law is currently pending before the Supreme Court of the State of New York (the "Court"), and the effectuation of the Commutation is subject to approval by the Court of the Agreement and certain related matters. Such approval is within the Court's sole discretion, and no assurance can be given that the Court will grant such approval or when it will be granted. If the Court grants such approval, Radian Asset will be required to make a commutation payment to FGIC in the approximate amount of $52.4 million once that approval becomes final in accordance with the Agreement, and the Commutation will become effective upon FGIC's receipt of such payment. The amount of this payment was determined primarily based on existing loss reserves and unearned premium reserves, and therefore is not expected to have a material impact on Radian's consolidated financial statements or Radian Asset's statutory capital position.
About Radian
Radian Group Inc. (NYSE: RDN), headquartered in Philadelphia, provides private mortgage insurance and related risk mitigation products and services to mortgage lenders nationwide through its principal operating subsidiary, Radian Guaranty Inc. These services help promote and preserve homeownership opportunities for homebuyers, while protecting lenders from default-related losses on residential first mortgages and facilitating the sale of low-downpayment mortgages in the secondary market.Check Out Our Best Services for Investors
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Avis A-List Blog Awards 2013
We are delighted to announce the winners of the 2013 Avis A-List Awards. They are as follows:
1st Prize – British Airways Travel Voucher worth £800
Fiona Maclean – London Unattached
2nd Prize – British Airways Travel Voucher worth £400
Laura Bonafoux – Laura the Explorer
3rd Prize – iPad Mini Wifi 32GB (RRP £349)
Lisa Eldridge – Girl About the Globe
The Voters Prize Draw – An Avis Car Rental Voucher worth £200
Shelley Sofier
Congratulations to all our winners and thank-you to all nominees and voters who participated in the awards.
The Awards were created to honour and appreciate the bloggers who help us all to discover new and exciting destinations. They take us along on every trip, share their experiences and inspire us to travel further more often, one post at a time.
Stay up to date with all things Avis by following us on Twitter and Facebook.
Don't forget to head over to the Avis website to find out more about car hire for your next trip!


text:
I found a white pill in my dresser drawer with Mylan A4 on it. Cannot remember what it was for. help>
Question is Closed | Report Mark as Favorite |
Answers (1)
24 Feb 2005
Round white double (+) scored tablet imprinted Mylan over A4 is 2 mg alprazolam.
More info: http://www.drugs.com/alprazolam.html
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text: Aug 15, 2013 22:40 "Club News" column for Aug. 6, 2013 "Club News" column for Aug. 6, 2013 Photo provided by RENEE WOODS -- Alpha Kappa Alpha -- Alpha Kappa Alpha Sorority's Nu Gamma Omega chapter delivered fans to Shiloh Missionary Baptist Church for the senior citizens on July 23. Shown are, from left, Isaiah Edah-Dike, Frankie Polland, Stella Jackson, Eula Smith, Cynthia Godfrey, Daraka Miles, Leslie Brown Vincent and Alvin Cavalier. Advocate story Aug. 15, 2013 Comments Sign language group celebrates third year Communicating Thru Sign celebrated its third year of teaching sign language classes in Baton Rouge on July 20. Founded by Carol Taylor-Elam, CTS was established to spread awareness about deaf culture and sign language and to bridge the communication gap between the deaf and hearing communities. CTS is offering free basic sign language classes every other Saturday from Aug. 10 through Oct. 19, from 10 a.m. to noon at the East Baton Rouge Parish Carver Branch Library. To register, email email@example.com or email@example.com. Plans are under way for the CTS 2014 "Praises on the Runway" summer fashion show. For information about CTS, visit communicatingthrusign.com. Compiled by George Morris Advocate staff writer The "Club News" column runs every Tuesday and Wednesday, and Friday as needed, in The Advocate. Club items and photographs should be submitted to "Club News," Advocate People section, P.O. Box 588, Baton Rouge, LA 70821, or emailed to email@example.com. Events should be submitted in a timely fashion. Photos will be returned if stamped, self-addressed envelopes are provided. By submitting photos to The Advocate, you agree that they can be published in any of The Advocate's print or digital publications. Get The Advocate in your inbox Subscribe


text:
Mergers and acquisitions involving Minnesota companies that were announced in the past week. Mergers and acquisitions involving Minnesota companies that were announced in the past week.
6/3 Rainbow Medical Ltd. Multiple investors Rainbow Medical, a provider of investment services to start-up Herzelia Pituach, Israel companies in the therapeutic medical devices market with an office in Minneapolis, received $50 million in funding from investors that include Fridley-based Medtronic Inc.
6/4 Medtox Scientific Inc. Laboratory Corp. of Laboratory Corp will acquire Medtox Scientific, a provider of New Brighton America Holdings laboratory testing services, for $242 million. Burlington, N.C.
6/4 Clutch & Transmission FleetPride Inc. FleetPride Inc., a distributor of heavy duty truck and trailer Service Inc. The Woodlands, Texas replacement parts, acquired a distributor of motor vehicle Roseville supplies, accessories, tools and equipment.
6/4 Identification division IdentiSys Inc. IdentiSys acquired the identification division of Claritus Inc., Lincoln, Neb. Eden Prairie which provides identification technology products and has offices in Nebraska and South Dakota.
6/5 OrangeSoda Inc. Deluxe Corp. Deluxe acquired an Internet marketing services company American Fork, Utah Shoreview specializing in search, mobile and social media campaign strategies for small business for $27.7 million.
6/7 Fairview Red Wing Mayo Clinic Mayo Clinic will acquire Fairview Red Wing Health Services, Health Services Rochester, Minn. which has facilities in Red Wing and Zumbrota, Minn., and Red Wing, Minn. Ellsworth, Wis.
6/7 Certain assets Cargill Inc. Cargill will acquire grain-handling assets in Colorado from Cheyenne Wells, Colo. Wayzata TradeOne, a grain storage company.


text:
Upon waking I need not have been concerned, one had been viewed 2,800 times (one "keyword") and clicked only three times, the other had been viewed 270 odd times ("two keywords" and clicked three times (two of which was me!).
As this is my first time, I have to admit to being slightly disapointed not with google just the numbers.
So my question is what sort of numbers do you get, we do not need specifics just the numbers.
CTR: 1.6 Avg. Pos: 2.6 CTR: 6.2 Avg. Pos: 4.6 CTR: 6.5 Avg. Pos: 2.0 CTR: 1.0 Avg. Pos: 4.5 CTR: 3.7 Avg. Pos: 2.2 CTR: 3.5 Avg. Pos: 1.5 CTR: 1.7 Avg. Pos: 4.2
I can't really see much of a direct corrolation relating position and CTR. If your budget doesn't permit you to bid up to the #1 spot don't worry too much about it. You can still garner decent CTR with a lower position.
If I were to group the keywords a bit better and target the ads a bit more I could probably increase some of those and eventually I will get around to it.
Mine are :
Camp - Imps - Clicks - CTR
BTW all these two campaigns are the only adword there, so am at the top.
KW1- Average Position 1.8, CTR 8.7% KW2- Average Position 1.4, CTR 9.6% KW3- Average Position 1.8, CTR 10.9%
10% is a lot of 10,000 impressions but not on only 100. Without knowing the impressions it is hard to gage.
I am sure most people here now roughly per day how many impressions their keywrods get.
7492636 33871 9970 42479 38421 6276 10817
Some of these campaigns are highly targeted and some are not as targeted.
The right copy and headline can make a big difference with AdWords. Try some different combinations and see what works best.


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My family and I are flying out the day before and I need hotels and ways to get there from the airport. Does anyone have suggestions that won't cost me a fortune? There are 3 of us. Thanks
We stay at a Disney resort and take magical express. I'm not sure how you're getting to port, so I don't know if this would work for you, but with only 3, you can book a value resort, so it would be inexpensive.
Sorry...I misunderstood. If you are looking for a hotel near port, I've never done that so I can't recommend. DCL transfers would run you $210, and hiring private transportation would be cheaper, probably around $190, depending on which company you choose. A rental car might be cheaper, but you'd have to factor in parking unless your hotel includes that. We take ME to a Disney resort, spend the night there, then take a private car on the day of the cruise. The car service can return you to the airport after the cruise, or back to the Disney resort if you wanted to spend another night and take ME back to the airport the next day. I'm sorry I can't be more help with the Port Canaveral hotels.
All the fly snooze and cruise I have looked into you have to put either $199 or $299 down but the catch is you have to review the Ron Jon Resort befor you get either a deposit of $45 or $145 back. But this includes round trip shuttle to and from the airport, the night in the hotel, and free transportation, also the Best Western you get free access to the water park at the resort. The room tax is only $8 and graduity is $8. What are all ya'lls opinions?
We stay at the MCO hyatt which is in the orlando airport for less than $200 depending on what kind of deal you get (priceline, stay certificates, aaa deals, etc). Then when we get up in the morning we usually do private transportation that costs in the $300 range. But for 3 people if you use the disney transportation bus for to and from then it is $70pp round trip which puts you at $210 for transportation, then add whichever hotel room you want. We like staying in the airport hotel cause we usually get a late flight in and we don't have to lug our luggage to a transport just to stay somewhere a night. Then in the morning you get up have some breakfast and just go down to the Disney transportation desk.


text: I was a bit put off this place by previous reviews,but my friend had stayed before and was keen to do so again. I was glad we decided to stay. Kampong Chhnang is very small and everywhere a tourist might go is easily accessible from the hotel. At $8 for a fan room it was good value, and the room... More


text: On Tuesday 17 August 2004 09:34, Rainer P. Feller wrote: > On Tue, 2004-08-17 at 07:15, michael wrote: > <snip much realy bad stuff> > whoa! bad things are happening in the world! Indeed, I thought South Africa was bad and understated in the media. The kind of stuff mentioned by the Michael should receive media attention but doesn't - ostrich phenomenon I guess (which is a myth btw - despite not being the brightest of creations, they don't avoid danger by putting their heads in the sand - they run, and if necessary kick with formidable force). Such things anger me greatly. During the days of apartheid SA was in the news daily here. Every little violation of human rights was dealt with in detail on the news and talk shows (and rightly IMO). After the victory by the ANC the only remaining news was praise for the ANC and Mandela (also rightly). However, no mention was made of the escalating crime and inept/corrupt beaurocracy until much later, and then only matter of factly and out of context. Ditto for HIV infections, although this is underreported in SA as well. I have friends in SA who work in rural hospitals who give a more accurate picture of the statistics than the media (similar stories of cues at hospitals - furthermore, most beds are occupied by aids patients. To make matters worse, patients have the right to refuse an aids test and do). Thankfully SA doesn't have the drug/mafia problem to the same degree that South American countries have (some African countries are cursed with diamonds which I suppose is similar). > I didn't thought that it is that bad in Brazil. > Why the Hell don't some parties cooperate? > Are they that different or don't they "just" want to > share the power? Ha - I came across a quote once which seems appropriate (can't remember who it was alas) "It's not that power corrupts, but that power attracts the corruptible". There are many problems as I see it - hang on, this could take a while. In no particular order: In my experience the "have-nots" aspire to western coca-cola, burger, cadillac and snappy-shoes culture. I suppose we should blame the marketeers and proponents of consumerism and the myth of sustainable growth here. The media predominantly show healthy people living ideal lives. Little mention is made of stress, heart disease, obesity ... Politicians use this, and somehow manage to raise the hopes of these people with promises. We tend to forget that "educated" and "enlightened" people are a small minority. The majority of the people on this planet are not really in a position to make a rational or informed decision regarding democracy (SA, being one of the more advanced countries in Africa still has voter education, which basically involves informing people that their votes are really secret - it's anyone's guess what democracy really means to them). People with a common heritage tend to stick together. The developing world consists predominantly of artificially created borders containing many peoples of different cultures. Oddly, people who have experienced poverty and manage to get out of it tend to turn their backs on the slums they came from. Perhaps they think "well I managed to get out of it so you can too" - I dunno. The champions of the poor generally come from an advantaged background and are sadly overshadowed by the champions of the rich. Family values are being (deliberately or not) degraded. The current system of democracy (4 year term -> elections) has the effect that parties make only short term policies that will "buy" votes. Confuscious says: "If you plan for 1 year, plant rice. If you plan for 10 years, plant trees. If you plan for 100 years, educate your children". I'd better stop. There's lots more, I think I've been in Holland too long as I seem to be losing track of some of these issues. > > And as far as I remember "nothing is off topic in lfs-chat" > Right - perhaps I'll open a new thread regarding my opinions on drug policies ... Got to start getting ready for nightshift now. Regards - Allard.


text:
It took BioPort several years to receive FDA approval, and during that time supplies of the vaccine ran low, necessitating suspension of the military's vaccination program. With the newly validated manufacturing process being used in a renovated facility, AVA will be produced under strict controls that are in accord with current FDA requirements. The report states that the newly produced vaccine is expected to have greater assurance of consistency than the vaccine produced at the time of its original licensure.
The committee emphasized the importance of continued and improved monitoring efforts to detect any adverse health effects caused by AVA and other vaccines. In addition, studies are needed to quantify and correlate protective levels of antibodies in animals with antibody levels in humans after full immunization. Direct tests of the efficacy of AVA are neither feasible nor ethical in humans. However, correlates of protection can be derived from studies that use animal models to test the efficacy of AVA, as well as new vaccines against anthrax. Both passive and active protection studies have important roles. The report stressed that production, testing, and licensure of a new vaccine requiring fewer doses and producing fewer local reactions is needed. The findings and recommendations of the report are presented in Appendix G. The full report can be found on the Internet at http://www.nap.edu/catalog/10310.html.
CDC has organized its Anthrax Vaccine Safety and Efficacy Research Plan into components of efficacy, safety, and acceptability. Accordingly, this report follows that structure to some extent. Chapter 2 provides background material about the disease known as anthrax, the licensed anthrax vaccine, and the questions regarding the efficacy and safety of AVA that prompted the congressional request for the CDC research program. Chapter 3 summarizes CDC's plan for the anthrax vaccine research program. In Chapter 4, the CDC research regarding the efficacy of AVA is described and the committee's findings and recommendations regarding this aspect of the research plan are presented. Chapter 5 describes the CDC research that is to address the safety of the anthrax vaccine, followed by the committee's discussion and evaluation. Chapter 6 reviews the research planned by CDC to address the acceptability of AVA and the committee's findings and recommendations about this research. In Chapter 7, the committee discusses and evaluates the completeness and appropriateness of the research plan as a whole.
CDC (Centers for Disease Control and Prevention). 2000. Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR (Morbidity and Mortality Weekly Report) 49(RR-15):1–20.
IOM (Institute of Medicine). 2001. CDC Anthrax Vaccine Safety and Efficacy Research Program. Interim Report. Washington, D.C.: National Academy Press.
IOM. 2002. Joellenbeck LM, Zwanziger LL, Durch JS, Strom BL, eds. The Anthrax Vaccine: Is It Safe? Does It Work? Washington, D.C.: National Academy Press.


text:
Perry: Politicians aren't perfect
POSTED: Saturday, June 15, 2013 - 4:07pm
UPDATED: Sunday, June 16, 2013 - 2:47pm
Gov. Rick Perry of Texas made light at a major gathering of conservatives on Saturday of his sometimes rocky 2012 run for the White House.
"I've learned a little bit about humility particularly on national television," he quipped to laughter at the Faith and Freedom Coalition's annual conference, which is one of the largest gatherings of social conservatives.
A few lines later in his faith-themed speech, Perry noted a critique of "St. Peter, whose mouth sometimes ran faster than his brain. Let me tell you, I can relate to Peter."
In a high-profile gaffe, Perry could not remember at a GOP primary debate the third of three federal agencies he planned to eliminate if elected. He later described it as a brain freeze and said in a CNN interview, "I bet there are a lot of Americans out there that would like to forget some agencies of government, too."
He made one on Saturday morning at this lower-profile event, confusing the country where four Americans, including a U.S. ambassador, were killed last September 11.
"Our administration won't make one phone call to save our men and women in embassies in Lebanon," he said, apparently referring to the attack in Benghazi, Libya, "but they'll monitor phone calls of 121 million Americans with one single judicial order. That bothers me."
But Perry, who has said he will announce this summer whether he will run for the governor again next year, with a decision on making a 2016 White House bid coming a bit later this year, told the half-full ballroom at a Washington hotel that nobody in politics is without flaws.
"The fact is God hadn't called the perfect to go into the arena of public service. He's called people just like you and just like me, nothing less than the example of our savior," he said.
He was the first speaker at the final day of the conference and spoke to a half-empty ball room. Nearly the full roster of prospective 2016 presidential candidates accepted invitations to speak at the conference.
Perry criticized the federal government's collection of internet and phone records, spoke out against the national debt and made a veiled criticism of the Obama administration's gun control efforts.
"I fear the trampling of the Second Amendment and the discarding of the Tenth Amendment. Every word in the Bill of Rights guarantees our freedom and not a single vowel of that should be ignored," he said.
To the point of freedom, Perry noted that he had this week signed into law religious freedom legislation which some in Texas called the "Merry Christmas bill."
"I have no idea why this is even required in America today," he said. "Expressions of faith I happen to think are very positive in the American society that we live in today."
Perry was followed on stage by Republican National Committee Chairman Reince Priebus. Former governor and vice presidential candidate Sarah Palin will close the conference Saturday afternoon.
Comments News Comments
And this smarmy little man is the granddaddy of all slick and shifty governors this state has ever had...along with 23 other vetoes this clown signed this past Friday, he vetoed the Lilly Ledbetter Fair Pay Act, a bill that was supported by the Texas legislature and would have ensured that women would receive equal pay for equal work on par with men...he also vetoed bills that would have tightened ethics laws and reined in the powers of university regents...you all got what you paid for, folk!!


text:
UPC Code: 738337883606
Monin Chocolate Chip Cookie bottles the true essence of a favorite childhood treat. Chocolate chip cookies are an American creation: Mrs. Ruth Wakefield of the Toll House Inn in Whitman, Mass. is credited with coming up with the first recipe in 1933. In 1997 the chocolate chip cookie was adopted as the official cookie of the State of Massachusetts. Nowadays, the cookies are baked in homes all across America.
Monin Chocolate Chip Cookie is one of our indulgent and nostalgic flavors, together with Monin Toasted Marshmallow, Caramel Apple, Peanut Butter and others.
Tasting Notes
� Distinct cookie samosa; true cookie and chocolate chip taste with hints of cookie dough
� Lattes, mochas, smoothies/shakes, hot cocoa, steamers/milk and cocktails


text:
Gallery: Announcing the Green Halloween Costume Contest Finalists!
Not only is Callie Williams' incredible peacock costume gorgeous – it's even got a functioning tail!
Halloween has come and gone and you might have already plowed through all of your collected treats, but luckily we've got some eye candy for you to feast on – the fabulous finalists from our Green Halloween Costume Contest! We were totally impressed (and in some cases terrified) by the flood of creative costumes we received and we've narrow them down to a select few that really showed what DIY eco costume making is all about. Now it's up to you to choose the lucky winners who will receive a sweet FEED Halloween bag filled with Yummy Earth Lollipops, a $100 gift certificate to the Inhabitatshop, and our fabulous Inhabitat t-shirts and tote bags!


text:
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- Report Post
- Posted: Fri, 04 May 2012 20:05:05 +0000
Birds and butterflies,
Rivers and mountains she creates,
But you'll never know,
The next move she'll make
The White Witch
You can try, But it is useless to ask why,
Cannot control her, She goes her own way,
She rules until the end of time, She gives and she takes
You can try, But it is useless to ask why,
Cannot control her, She goes her own way,
She rules until the end of time, She gives and she takes
- Treasa never got angry or upset with Fendrel. She knew he was only excited, and the closer she got to the end of the pregnancy, the more excited she got too. The child's kicking had gotten old for her by now, and there were times where she wished the girl would give it a rest, but the look on Fendrel's face each time she told him was worth the girl rearranging things inside her. His mother and Narcissa weren't the only ones that'd made things for the baby, Treasa had as well, and she'd even taken to sitting down with Elena to make the clothes. Someone had to teach her what she was doing with such tiny clothes, and while she was grateful for Ema and Narcissa, Elena was family now, and she needed to get to know her, so she had been.
Treasa always held her breath on the way down steps. She could get up them fine, but getting down them, it was too easy to miss one, and Fendrel had already caught her on more than one occasion when she miscalculated where to put her foot down. When they reached the ground, Treasa let her breath out, watching as the rest of the group greeted Nathaniel and those he'd come with came down from their horses. It didn't bother her that Nathaniel's arm was in a sling, he could have come back a lot worse of injury, or Gods forbid, in a shroud. When Nathaniel saw her, he cracked a smile and said, Gods be good you're-" Narcissa quickly clasped a hand over his mouth. Treasa had already made Marcus regret calling her huge, and Narcissa wanted more than five minutes with the husband she hadn't seen in months.
Nathaniel moved Narcissa's hand from his mouth gently. "I was going to say radiant, Narcissa." He kissed her on the forehead, then came over and hugged Treasa tightly before looking at Fendrel. "I'm glad to see me alive too! Bloody idiots aren't nearly as organized as they think! Turns out we didn't need you in Graymore! Maeve here's a good enough shot she was able to cover me just fine!" Narcissa came over, peeking into the sling at his arm. "Relax, my love, that's my shield arm." Narcissa didn't say anything, only nodded in understanding when he reminded her that that was his shield arm.
With every breath, And all the choices that we make,
We are only passing through on her way, I find my strength,
Believing that their souls live on, Until the end of time, I'll carry them with me
We are only passing through on her way, I find my strength,
Believing that their souls live on, Until the end of time, I'll carry them with me


text:
Founded in 1787, Marylebone Cricket Club is the most active and famous cricket club in the world and owner of Lord's Cricket Ground - the Home of Cricket.
© Copyright 2015
Home News Latest News Lord's
The returning Chris Gayle is the star attraction for the West Indians when they take on Middlesex at Lord's on Wednesday 13 June.
Join Lord's TV and Oxford MCC Universities captain Ben Williams as he guides you through what it's like to be a cricketer at one of the six MCCUs.
Lord's will host an Investec Test match between England and New Zealand in May 2013, originally scheduled to be staged in Cardiff.
Middlesex take on the surprise package of the 2012 CB40 season so far, the Netherlands, at Lord's on Sunday 3 June.
Middlesex will be boosted by the presence of England duo Steven Finn and Eoin Morgan when they take on Sussex in the LV= County Championship first division at Lord's, starting on Wednesday 30 May.
In his latest blog for Lords.org, Middlesex's John Simpson discusses his side's fighting character, losing Lasith Malinga and gaining Eoin Morgan.
In his latest blog, exclusively for Lords.org, MCC World Cricket committee Chairman Mike Brearley welcomes his four new Members, and thanks those leaving.
Lord's TV joined thousands of spectators on the outfield at the Home of Cricket on day five of the England v West Indies Test, after MCC permitted perambulation.
Cricket, more than most sports, values statistics and history in almost equal measure, particularly in Test cricket.
Lord's TV takes you behind the scenes as the Home of Cricket prepares for the Test match between England and the West Indies.
Switchboard: 020 7616 8500
Ticket Office: 020 7432 1000
Founded in 1787, Marylebone Cricket Club is the most active and famous cricket club in the world and owner of Lord's Cricket Ground - the Home of Cricket.
© Copyright 2015


text:
quote:White American from my experience
quote:Rather than someone just saying "indian women are freaks in bed" or the like, the person should also list the nationalities he has experienced with, or else the survey answers don't mean much.
quote:Hands down Latino
quote: Israelis SUCK!
quote: Really? I've heard they have a permanently fatalist mentality which makes them film-noir kinky.


text: Pope calls for worshipers to remember 'essence' of ChristmasBy the CNN Wire StaffUpdated 5:55 AM ET, Thu March 29, 2012Pope Benedict XVI told the faithful at Midnight Mass that "we must dismount from the high horse of our 'enlightened' reason."Story highlightsBenedict deplores the "superficial glitter of this season"A nativity scene is unveiled in St. Peter's SquareThe pontiff will deliver his annual Christmas message SundayPope Benedict XVI presided over Midnight Mass on Christmas Eve, delivering a homily that focused on the "essence" of the holiday rather than the "commercial celebration" it has become."Today Christmas has become a commercial celebration, whose bright lights hide the mystery of God's humility, which in turn calls us to humility and simplicity," the pope said after recalling the story of Christmas. "Let us ask the Lord to help us see through the superficial glitter of this season, and to discover behind it the child in the stable in Bethlehem, so as to find true joy and true light."The 84-year-old pope, presiding over his seventh mass as pontiff, also conjured up an image of the Church of the Nativity in Bethlehem, explaining that visitors must bend down to enter its low opening, drawing a tie to what followers of Christ must do to find their faith."If we want to find the God who appeared as a child, then we must dismount from the high horse of our 'enlightened' reason," he said. "... In this spirit let us celebrate the liturgy of the holy night, let us strip away our fixation on what is material, on what can be measured and grasped."The pope started off Saturday's celebrations by lighting a peace candle in the window of his study during the unveiling of a larger-than-life nativity scene in St. Peter's Square. The scene, at 23 feet high and 82 feet wide, is dedicated to the Virgin Mary, according to the Vatican's news agency.Just WatchedPope delivers Christmas Eve MassreplayMore Videos ...Pope delivers Christmas Eve Mass 01:06PLAY VIDEOJust WatchedPope Benedict XVI's Mass in HavanareplayMore Videos ...Pope Benedict XVI's Mass in Havana 02:20PLAY VIDEOThousands poured into the square and St. Peter's Basilica ahead of the Midnight Mass, which was actually held at 10 p.m. this year. Before his homily, the aging pontiff rode down St. Peter's long aisle on a wheeled platform, waving to worshipers.Benedict will follow the Christmas Eve celebrations with his annual Christmas message "To the City and To the World" -- Urbi et Orbi in Latin -- on Sunday.More from europeWhere does Russia's opposition go from here?Slain Kremlin critic Nemtsov thought fame offered protectionOfficials: 33 bodies recovered from eastern Ukraine mine disasterPowered by Livefyre


text:
Our FS 4800 D is a very versatile diesel walk-behind flat saw ideal for asphalt and concrete cutting. Suitable for mid-sized jobs on airports and highways, as well as service work and smaller jobs that require high production rates. Cutting depth up to 381 mm. Available in front or rear pivot configurations, where the rear pivot saw is recommended when high manoeuvrability is essential.
Well-balanced design with a low profile gives you high visibility and makes it easy to handle and transport.
Patent pending electric tracking system lets operator make tracking adjustments while cutting.
The handles (patent pending) are easily adjusted, so the user can find the most comfortable working position.
The IntelliSeal blade spindle (patent pending) has four sealed radial bearings for strength and durability. Allows for a minimum of 500 hour maintenance intervals and a 1000 hour limited warranty.
Including temperature gauge, voltmeter, tachometer and blade clutch switch.
The patented transmission with right-angle gearbox minimises vibrations and increases efficiency at the blade spindle.
The tachometer/bladeshaft rpm gauge on the FS 6600 D and FS 4800 D indicates engine rpm or bladeshaft rpm. Bladeshaft rpm range is colour coded to gear selection position.
On the FS 6600 D and FS 4800 D, the gearbox shift lever is used to change the output speed of the engine gearbox. Three speeds and two neutral positions are standard. The gearbox shift lever positions are colour-coded to match the blade diameter and the bladeshaft rpm decal located on the control panel. Gearbox detent locks the gearbox shift lever in position.
Dual light kit
Catalytic converter kit, for outdoor use only.
Blade shaft belts
Spare parts
Blade collars
Front wheel
Engine oil filter
Fuel filter
Find your local Husqvarna salesman or dealer using the dealer locator.


text: 4.0L V6 engines may develop a buzzing type noise from the upper engine area at about 1900 RPM, under light acceleration. Our technicians tell us this can be caused by loose ignition coil bracket bolts. Coil bracket bolts should be checked for the correct torque before further diagnoses is performed.