text:
13 Mar 2008: Tallóczy Z, Martinez J, Joset D, Ray Y, Gácser A, et al. (2008) Correction: Methamphetamine Inhibits Antigen Processing, Presentation, and Phagocytosis. PLOS Pathogens 4(3): 10.1371/annotation/bd02ad26-a081-4c61-88c2-ebda285b8bca. https://doi.org/10.1371/annotation/bd02ad26-a081-4c61-88c2-ebda285b8bca View correction
Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal–lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.
Author Summary
There is a new population of HIV+ men who are developing AIDS over months instead of years as typical. It has recently become popular among gay and bisexual men to consume very high levels of Meth. Unsafe sex together with Meth abuse has been suspected to lead to rapid disease progression. While studies show exacerbated AIDS symptoms and disease progression in HIV+ Meth abusers, the molecular mechanism is yet unknown. It was postulated, yet unproven, that the rapid disease progression might be due to a mutant "superstrain" of HIV that was extremely virulent. It was also assumed that the effects of the drug on behavior may lead to unsafe sex, although this would not explain the more rapid time course of the disease. We now demonstrate the first direct evidence that Meth is an immunosuppressive agent, and that the molecular mechanism of this immunosuppression is due to the collapse of acidic organelle pH in cells of the immune system, inhibiting the functions of antigen presentation, as well as phagocytosis. These effects compromise the immune response to opportunistic infections and HIV. These findings could have a major impact on public health, as there are over 35 million Meth abusers worldwide
Citation: Tallóczy Z, Martinez J, Joset D, Ray Y, Gácser A, Toussi S, et al. (2008) Methamphetamine Inhibits Antigen Processing, Presentation, and Phagocytosis. PLoS Pathog 4(2): e28. https://doi.org/10.1371/journal.ppat.0040028
Editor: Susan Ross, University of Pennsylvania School of Medicine, United States of America
Received: August 24, 2007; Accepted: December 28, 2007; Published: February 15, 2008
Copyright: © 2008 Tallóczy 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 Institute on Drug Abuse and the Parkinson's Disease Foundation (DS), the Einstein/Montefiore Medical Center for AIDS Research (LS), National Institutes of Health (LS) and Irene Diamond Professorships in Immunology (LS).
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: Ca, Candida albicans ; Cn, Cryptococcus neoformans ; Clq, chloroquine; [E(IgG)], IgG-antibody coated erythrocytes; GFP-LC3, green fluorescent protein fused to microtubule-associated protein 1 light chain 3; LAMP-1, lysosome-associated membrane protein 1; Meth, methamphetamine
Chronic methamphetamine (Meth) abuse has reached epidemic proportion throughout the United States, where a 2003 survey indicated that approximately 5% of the population over 12 years of age has tried Meth and the rate of treatment admissions for primary Meth abuse increased over 3-fold (The DASIS Report, http://www.oas.samhsa.gov/2k6/methTX/methTX.htm) [1,2]. In particular, among gay and bisexual men [3] Meth it is associated with high-risk sexual behavior, HIV viral infection, and a high incidence of AIDS [4]. Meth exacerbates AIDS pathology, including cognitive deficits [5,6], and is strongly suspected to inhibit normal immunological response to secondary infections such as hepatitis C, which is prevalent in those who smoke or take Meth intranasally [7]. It has recently been suggested that Meth could contribute to a particularly rapid progression of AIDS in individuals exposed to a strain of HIV that is resistant to drug treatment [8,9]. Indeed, animal studies clearly demonstrate that Meth suppresses both innate and adaptive immunity [10,11], enhances cytokine production in combination with HIV TAT protein [12], and alters gene expression in cells of the immune system [13]. However, the molecular basis for Meth's immune suppression is unknown. Here we have examined the relationship between Meth and the impairment of specific immune cell functions.
In the clinical setting, Meth abusing individuals who present with opportunistic infections possess high blood and tissue levels of the drug. Meth is generally self-administered in this population in binges of 3–4 grams ingested over a six day interval [14], with an initial ingestion of ∼0.5 gram (Judith Rabkin, Columbia University, personal communication) and a total mean level of 2.2 grams ingested during the first day [15]. Such high levels of administration result in a blood concentration of ∼10–50 μM Meth, and levels in the hundreds of micromolar range in organs including brain and the spleen [16] (see Results).
Pathogens are processed and displayed by antigen presenting cells (APCs) for T cell recognition. Antigen presentation in tissue resident macrophages, as well as dendritic cells, involves fluid phase or receptor-mediated endocytosis followed by fusion of the phagosome with specialized lysosomes known as MHC class II compartments (MIIC) [17]. The foreign antigens are partially degraded by lysosomal hydrolases and the resulting peptides are loaded on MHC class II molecules and transported to the cell membrane to be presented to T cells. Both pathogen transport to the MIIC and its degradation into immunogenic peptides are functions that require an acidic endosomal pH. Endogenous antigens and viruses can be delivered to the MIIC upon autophagosome fusion [18]. Autophagy also mediates single-stranded RNA virus detection and consequent interferon-α release in plasmacytoid dendritic cells activating anti-viral cellular defense mechanisms in uninfected cells [19]. Further evidence shows the direct role of autophagosomal-lysosomal degradation in elimination of intracellular HSV-1 viral particles [20] and that the HSV-1 has alternative methods to counteract cellular autophagy [21].
In this study, we have identified a novel molecular mechanism that explains the immunosuppressive effects of Meth via the alkalization of acidic organelles in dendritic cells and macrophages that are critical for the immunological function of these APCs.
Estimates of Pharmacological Meth Levels in Humans
To model effects of Meth on the immune system, we estimated Meth levels in drug abusers. Meth is self-administered intravenously, by nasal inhalation, anally, and orally, in typical doses of 250–500 mg by occasional users to levels as high as 1 g by chronic abusers (personal communication, Perry N. Halkitis, New York University). Meth blood levels measured in individuals detained by police in California were 2.0 μM on average but as high as 11.1 μM [22]. Controlled studies indicate that a single 260 mg dose reaches a level of 7.5 μM [22]. Thus, a single dose of 260 mg – 1 g would be expected to produce 7.5 – 28.8 μM blood Meth levels.
The abusers however tend to self-administer METH in binges, and as the drug exhibits a half-life of 11.4 - 12 h [23,24], this leads to higher levels. Recently published studies modeling binge pattern of use in individuals show that after the fourth administration of 260 mg during a single day produces maximum blood levels of 17 μM, reaching 20 μM on the second day of such a binge [22]. Thus, binge doses of 260 mg – 1 g would produce 17 - 80 μM blood Meth levels. The estimates appear consistent with blood levels detected after fatalities [25–27], ranging as high as 84 μM for an individual for whom Meth intoxication was determined as the cause of death ∼16 hours after ingestion.
It is also important to estimate how Meth is distributed from blood to other tissues involved in immune response, particularly the spleen, which houses high numbers of dendritic cells. Tissue-to-serum Meth ratios in rat are: brain, 9.7; kidney, 35.3; spleen, 14.3 [16]. Thus, relevant levels in spleen, the organ critical for immune response, after administration of 250 mg – 1 g as a single dose is 100 – 400 μM, and during binges between 240 – 1144 μM.
Meth Induces Rapid Alkalization of Acidic Organelles and Changes in Endosomal Morphology
Dendritic cell MIIC processing organelles are characterized by acidic pH [28], a limiting membrane enclosing internal vesicles or lamellae [17], the presence of proteases [29], internal expression of LAMP [30] and MHC II proteins [31]. The morphological and functional integrity of the MIIC depends on the maintenance of an acidic pH [32] due to the action of the V-ATPase, which may also regulate transport from early to late endosomes and lysosomes [33]. Meth and its metabolite, amphetamine, are membranophilic weak bases that collapse intracellular organelle pH gradients in neurons [34,35]. We therefore tested whether Meth collapsed intracellular organelle pH gradients in dendritic cells by monitoring quenching of acridine orange, a weak base vital dye that accumulates in acidic organelles including endosomes and lysosomes [35]. We found that Meth concentrations of 50 μM or higher rapidly collapsed acidic organelle pH gradients in dendritic cells (Figure 1A–1B). LysoSensor Yellow/Blue ratiometry is a generally accepted method to measure organellar pH in live cells, and was used to measure average pH in acidic organelles using LysoSensor Yellow/Blue fluorescent dye. After 10 min treatment with Meth (50 or 100 μM) or chloroquine (10 μM) acidic organellar pH was elevated (pH 6.5, 6.9 and 6.4 respectively) significantly above the levels in untreated control cells (pH 4.8) (Figure 1C). As predicted with alkalizing agents, Meth also disrupted MIIC structure [36], producing large organelles (> 1 μm diameter) devoid of internal vesicles (right panels) with LAMP-1 and MHC II staining confined to the limiting membrane (Figure 1D and 1E). Chloroquine (Clq) [10 or 20 μM], another weak base was used as a control showed similar effects to Meth as described above (Figure 1A–1E). At the used concentrations (20 and 100 μM) Meth did not affect cell viability as determined by flow cytometric analysis (Figure S1).
(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25–35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)
(B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25–35, mean ± SEM, Student's two-tailed t-Test).
(C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30–90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)
(D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm. (E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.
Meth Inhibits Endosomal Antigen Processing and MHC II Restricted Antigen Presentation
To investigate whether Meth-induced endosomal alkalization blocks antigen processing by impairing dendritic cell lysosomal proteolytic degradation of foreign proteins, we exposed cells to the fluorescently labeled MHC II antigens [bovine serum albumin (BSA), casein, and ovalbumin (OVA)], and measured the degradation of each protein by western blot. In untreated cells, each antigen was proteolytically degraded, while antigen degradation was blocked in Meth or Clq treated cells (Figure 2A and 2B). Meth (10, 50, 100 μM) and Clq (10, 20 μM) effectively inhibited processing of antigens previously taken up by dendritic cells (Figure 2A). Control experiments showed that Meth did not block endocytosis of 2 μm fluorescent dextran beads by dendritic cells (Figure S2), indicating that the drug does not inhibit nonspecific phagocytosis [37] .
(A) Western blots of bone marrow derived dendritic cells which phagocytosed FITC-labeled BSA, casein or OVA overnight, and exposed to Clq or Meth. Unprocessed antigens are quantified using anti-FITC mAb.
(B) Densitometric analysis of western blots for the amount of unprocessed intracellular antigen. Representative of two to three independent experiments.
(C) β-hexosaminidase activity measured in sub-cellular fractions to identify lysosomes and late endosomes.
(D) Western blot using LAMP-1 and TrfR mAb on lysosomal, as well as early and late endosomal fractions.
(E) Western blot analysis of the above pulled fractions from untreated, Clq- or Meth-treated dendritic cells using anti-Fitc mAb.
(F) Immunoprecipitation of invariant chain in pulse-chase experiments. Full length invariant chain (p31) and degradation products (p25/28 and p/10) are indicated.
(G) T cell proliferative response to bone marrow derived dendritic cells cultured with OVA protein (0, 3, 10, and 30 μM) in the absence (Ctr •) or presence of Meth (10 ○, 50 □, 100 μM ▵) or Clq (5 ▪ , 10 μM ▴) for 6 h, and cultured with T cells for 3 d. [3H]-Thymidine incorporation was used to assay T cell proliferation. (Statistics n = 4, mean ± SEM, **p < 0.001, two-tailed ANOVA.)
(H) T cell proliferative response to bone marrow derived dendritic cells cultured with OVA peptide 323–339 (0, 3, 10, and 30 μM) in the absence (Ctr •) or presence of Meth (10 ○, 50 □, 100 μM ▵) or Clq (5 ▪ , 10 μM ▴) for 6 h, and cultured with T cells for 3 d.
To determine the stage at which Meth compromised post-endocytic proteolytic antigen processing, we prepared fractions of early and late endosomes and lysosomes from dendritic cells exposed to BSA and casein antigens. Each fraction was examined for β−hexosaminidase to identify lysosomes and late endosomes, the transferrin receptor (TrfR) to identify early endosomes, and LAMP-1 to identify late endosomes and lysosomes (Figure 2C and 2D). No detectable antigen remained in early endosomes under any of these conditions. There were, however, much higher levels of BSA and casein in lysosomes and late endosomes of Meth- and Clq-treated cells than in controls. In particular, casein was completely degraded in untreated cells but relatively unprocessed in dendritic cells treated with Meth or Clq (Figure 2E). Thus, Meth inhibited antigen proteolysis within late endosomal/lysosomal compartments. Similarly, processing of invariant chain was compromized after Meth treatment as indicated by increased levels of p25/28 and p10 fragments during the chase time-point (Figure 2F).
MHC II antigen presentation stimulates T cell proliferation, providing a means to measure effects of Meth on antigen presentation. We prepared cultures of immature bone marrow-derived dendritic cells and splenic purified T cells, both from OTII transgenic mice [38]. Using OVA as an antigen, we assayed cellular proliferation of T cells by radiolabeled thymidine uptake and incorporation into DNA. At all levels tested, Meth decreased the T cell proliferative response to the intact antigen (Figure 2G) but not the pre-processed OVA-323–339 peptide (Figure 2H).
Autophagosome Accumulation Is Induced by Meth
In addition to blocking lysosomal antigen degradation, Meth could disrupt processing and presentation of antigens by inhibiting autophagosome formation, thereby halting antigen delivery to MIIC. To test this possibility, we prepared dendritic cells from a transgenic mouse expressing a GFP-fused autophagosome-associated protein LC3 (GFP-LC3) which has been used as an in vivo autophagosomal marker [39]. Consistent with previous reports in neurons [40], 50–500 μM Meth induced autophagosome accumulation in dendritic cells (Figure 3A–3C). Our data show that Meth does not block autophagosome formation, but rather impairs lysosomal-autophagosomal degradation, resulting in the accumulation of autophagosomes, and impaired intracellular antigen proteolysis. Together with the evidence from endosomal/lysosomal fractions (Figure 2C–2E), these results strongly suggest that Meth inhibits degradative antigen processing by disrupting pH gradients.
(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300–400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001).
(B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300–400 cells, mean ± SEM, ***p < 0.0001).
(C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.
Meth Alkalizes Acidic Organelles and Induces Autophagosome Formation in Macrophages
As with dendritic cells, we also found that Meth and Clq effectively collapsed the intracellular pH gradients within macrophages (Figure S3A–S3C) and blocked autophagosome degradation, resulting in the accumulation of GFP-LC3 labeled autophagosomes (Figure S4A–S4D). Similar results were obtained with the fluorescent dye monodansylcadaverine, a lipophilic weak base that accumulates in lysosomes and autophagosomes (Figure S4E).
Meth and Clq Inhibit Phagocytosis in Macrophages
Since Meth users can also present with bacterial infections [41], we examined whether Meth disrupts macrophage phagocytosis, a primary mechanism for clearance of these extracellular pathogens. Murine peritoneal-derived macrophages were incubated in the presence or absence of Meth. After 2 h, IgG-antibody coated erythrocytes [E(IgG)] were added to each of the conditions and the number of ingested erythrocytes counted. We found that Meth (50 and 250 μM) inhibited E(IgG) phagocytosis (Figure 4A and 4B) by 20 and 45%, respectively. Meth did not inhibit the receptor-independent endocytosis of Lucifer Yellow (Table S1). Similar effects were observed with Clq (Figure 4A and 4B) These results demonstrate that Meth inhibits Fcγ-mediated phagocytosis in macrophages and are consistent with the observations that the macrophage Fcγ receptors are continuously recycled between phagosomes and the plasma membrane [42], a process that requires appropriate acidification of secretory vesicles and tubules for trafficking [43]. These results demonstrate that Meth inhibits Fcγ-mediated phagocytosis in macrophages.
(A) Cells were incubated in indicated concentrations of Meth or Clq for 2 h, and then IgG coated erythrocytes were added. Extracellular uningested EIgG were lysed and removed. Phagocytic index (PI) was quantified and inhibition is indicated as percent of control. Data were collected from four to seven experiments (n = 300 cells, mean ± SEM, ***p < 0.0001, two-tailed ANOVA).
(B) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment. Arrows denote phagocytosed erythrocytes. Scale bar, 10 μm.
(C, D) J774.16 cells were exposed to PBS, chloroquine, or methamphetamine for 2 h followed by incubation with Cn or Ca. The phagocytic indices (ratio of intracellular yeast to the number of macrophages counted) were determined after 1 h or 30 min for Cn and Ca, respectively (C). (Statistics n = 300, mean ± SEM, *p < 0.05, two-tailed ANOVA.) CFU determinations were preformed after 24 h incubations (D). The values are the mean and standard deviation from determinations made in triplicate wells (n = 300 cells, mean ± SEM, *p < 0.05, two-tailed ANOVA.). Experiments were repeated three times with similar results.
Meth and Clq Inhibit Macrophage Phagocytosis and Intracellular Replication of Candida albicans and Cryptococcus neoformans
As a next step we analyzed the effects of Meth on Candida albicans (Ca) and Cryptococcus neoformans (Cn) phagocytosis and killing by murine macrophages since these two organisms are the most commonly isolated fungi in individuals infected with HIV [44]. Clq and Meth (10 and 50 μM) inhibited phagocytosis of Ca and Cn by macrophages by 40% (Figure 4C). Moreover, Meth enhanced the proliferation of fungi within macrophages (Figure 4D), indicating that intracellular replication of both yeast was facilitated by Meth. In contrast, Clq had no significant, or slightly reducing, effect on Ca (p= 0.056) and Cn (p = 0.060) CFU numbers in macrophages. Control experiments showed that in the absence of macrophages, Ca and Cn proliferation was unaffected by the addition of Clq or Meth to the BHI medium (data not shown).
Meth-Treated HIV-Infected Macrophages Exhibit Increased p24 Secretion
To further examine why chronic Meth abuse has been recently associated with the rapid development of immune deficiency among gay and bisexual men [3], we studied the effect of Meth on HIV proliferation in macrophages from HIV-transgenic mice. These JR-CSF/huCycT1 double transgenic mice express HIV-1 JR-CSF, which is a full length R5 HIV-1 provirus regulated by the endogenous HIV-1 LTR, as well as the human cyclin T1 controlled by a murine CD4 expression cassette [45]. These mice have constitutive HIV production in CD4 T lymphocytes and monocytes. GM-CSF differentiated bone marrow cells were either left untreated or treated with a range of Meth levels (10, 50, 150 μM) or NH4Cl (10 mM), another weak base, for 7 days. Cell supernatants were then collected and p24, a secreted HIV-specific protein, was quantified by Elisa. Cells from JR-CSF/huCyc T1 mice treated with Meth for 7 or 9 days exhibited a 30–60% increase (Figure S5A) of p24 antigen production. Clq does not provide a positive control, since its well-established inhibition of HIV production is probably due to inhibition of viral capsid protein glycosylation in the Golgi [46]. Thus, in this experiment NH4Cl was used as positive control (Figure S5A).
To determine whether HIV replication was affected by Meth in vivo, we studied the effect of Meth on HIV virus proliferation in the JR-CSF/huCycT1 double transgenic mice [45]. Mice were treated with increasing concentration of Meth over a 7 day-period. One group of animals received 5 mg/kg of Meth at day 0, 2 and 4 and was sacrificed at day 6 (low Meth). Another group received 6 mg/kg at day 0; 7 mg/kg at day 2 and 7.5 mg/kg at day 4 (high Meth) and was also sacrificed at day 6. Copy number of HIV-1 RNA was quantified in the serum of each mouse by RT-PCR using primers spanning the highly conserved region of the HIV-1 gag gene. No statistically significant differences were observed between the untreated and the Meth-treated mice (Figure S5B). These data suggest that Meth treated HIV-1 transgenic mice do not exhibit an increase in HIV viral load.
We find that the widely abused addictive psychostimulant, Meth, at pharmacologically relevant levels acts as an immunosuppressive agent, due to its inhibition of endosomal acidification. These actions result in Meth's inhibition of antigen presentation and phagocytosis. Maintenance of low endosomal and lysosomal pH serves many functions, including regulation of protein degradation, pathogen inactivation, and regulation of the amount of several surface receptors. All of these functions require active transport via the endocytic pathway and fusion with lysosomal compartments.
First, via alkalization, METH-inhibited lysosomal-autophagosomal degradative function for both exogenously and endogenously internalized antigens resulting in accumulation of proteins entering the endocytic pathway through phagocytosis, as well as autophagic vacuoles. Chaperone-mediated autophagy and macroautophagy in lysosomes have been described as major pathways for endogenous antigen processing in MHC class II compartments [18,47,48] and as a means to directly degrade intracellular virus particles [20,21]. Thus, through its alkalizing effect, Meth blocks normal antigen processing and presentation. Progression of internalized antigens along the endocytic pathway rely on the progressive maturation of early endosomes into late endosomes and ultimately to lysosomes. Maintenance of an acidic internal pH and a pH gradient in these compartments is important for the cargo progression. Endosomal acidification is accomplished by H+ transport across the endosomal limiting membrane by the proton pump vacuolar ATPase (V-ATPase). The recent discovery that V-ATPase interacts with components of the endocytic transport machinery indicates that V-ATPase is also a pH sensor that regulates early to late endosomal transport [33]. This would explain why endosomal alkalization by Meth not only disrupts antigen processing but phagocytosis and cargo progression along the endosomal pathway.
Second, endosomal alkalization by Meth inhibited Ca and Cn phagocytosis and killing by macrophages. These effects are expected to be particularly devastating in AIDS-related disorders, since Ca and Cn are the two most commonly isolated fungi from sterile body fluids obtained from HIV infected individuals [44]. Also, these results could explain recent reports of rapid insurgence of AIDS in Meth-addicted individuals soon after infection with HIV. Thus, Meth also blocks pathogen killing by macrophages. Exposure to Clq and other weak bases was already been shown to inhibit growth of Cn in macrophages [49,50] even though Clq has no direct toxicity to Cn [50]. Not all basic compounds are equivalent in their fungicidal activity; for example, ammonium chloride also enhances the anti-cryptococcal activity of macrophages, yet the potency of ammonium chloride is less than that of Clq, which may be a result of the ability of ammonium chloride to inhibit phagolysosomal fusion [51]. Hence, Meth apparently has complex effects on macrophages that result in an intracellular milieu that enables the replication of the examined pathogenic fungi.
Third, even though an increase in p24 secretion has been observed in Meth-treated dendritic cells and macrophages, a direct effect of Meth on HIV viral load could not be demonstrated. It is likely that viral proteins enter late endosomal and lysosomal compartments independently of viral assembly, and due to inhibition of processing, more viral proteins are secreted in the extracellular milieu. Within the infected macrophages, HIV has been previously shown to assemble in compartments with characteristics of multivesicular late endosomes (CD63, Lamp-1, CD81 and CD82 positive) [52]. It now appears that the HIV particles present in multivesicular endosomes are the results of endocytosis, and the site of viral assembly is at the invagination of the plasma membrane particularly enriched in tetraspannin proteins [53]. These results would explain why, by compromising endosomal pH, an increase in HIV viral load has not been observed.
In conclusion, the immunosuppressive effects of Meth are consistent with reports that Meth-treated mice demonstrate decreased immunity [10,11]. To be noted is that even though there is a linear concentration-dependent response between Meth concentration and pH disruption such an effect is less evident at the biological level. In all assays (antigen processing, Ca or Cn killing and p24 production) different Meth dosages behave very similarly. Likely a small disruption in the endosomal pH is sufficient to alter the microenvironment and endosomal-related functions. This is consistent with the effects of low concentrations of Meth tested on lysosomal pH in Figure 1C, with alkalinization of > 1 pH unit, which would effectively inhibit lysosomal protease activity.
The collapse of endosomal pH by Meth and the resulting decrease of normal immune response provide an explanation for the compromised immunity and exacerbate infections occurring in Meth abusers. In fact, Meth is strongly suspected to more dramatically inhibit normal immune responses than other drugs of abuse since Meth users often present with skin lesion and "Meth mouth", a devastating periodontal disease (http://www.drugfree.org/) [54] and [1,2]. A similar immunosuppressive activity has also been shown for chloroquine, also a well known inhibitor of endosomal acidification [36].
In particular, Meth immunosuppression may underlie the mechanism of the recently reported extremely rapid development of immune deficiency, with devastating effects in AIDS-related disorders in Meth abusers that had contracted HIV. In particular, there is evidence suggesting the presence of a new population of HIV+ positive men who are developing AIDS over months rather than over 10 or more years as is typical. The most widely reported individual was documented by Dr. Martin Markowitz of the Aaron Diamond AIDS Research Center in New York in 2004. In this individual, a gay man who was also a Methabuser tested HIV negative in May, 2003, then likely contracted HIV during unprotected sex in mid-October 2004, displayed acute retroviral syndrome in November, 2004 and 3-drug-class-resistant HIV-1 (3DCR HIV) with apparently rapid progression to AIDS by December, 2004 (CDC, MMWR July 28, 2006/55(29);793–796). Meth self-administration by HIV+ individuals during the acquisition of sexually transmitted pathogens appears likely to interfere with immunological resistance and lead to AIDS progression.
Materials and Methods
Additional detailed methods used in this paper can be found in Protocol S1.
Primary cell culture.
Mouse femur hematopoietic stem cells from bone marrow were harvested from the hind legs of 8 to 12-week old male wild-type C57BL/6J (The Jackson Laboratory, Bar Harbor, Maine) or GFP-LC3 transgenic mice [39], and plated at 2 × 106 cells/ml density in DMEM supplemented with 10% FBS, 1x non-essential amino acids (Gibco, Carlsbad, California), 2 mM L-glutamine, 1 mM sodium pyruvate and 20 mM HEPES. For differentiation to dendritic cells or macrophages, 10 ng/ml of recombinant mouse GM-CSF (Biosource, Carlsbad, California) or 10 ng/ml of recombinant mouse M-CSF (R&D Systems, Minneapolis, MN) was added to media, respectively. Cells were fed every 2 days with fresh DMEM containing the appropriate macrophage colony stimulating factor. Cells were trypsinized after 8 days and, unless otherwise noted, plated at 4 × 105 cells/cm2 density to be used for experiments the following day.
Resident mouse peritoneal macrophages were isolated from 8 to 12 week-old female wild-type C57BL/6J as described [55] and plated on 12 mm-diameter glass coverslips in 24-well tissue culture plates at 3 × 105 cells/well density in RPMI media supplemented with 10% FBS with streptomycin and penicillin. Non-adherent cells were removed by washing 2 h after plating. The remaining adherent cells were over 95% macrophages as assessed by esterase staining [55]. Cells were incubated overnight prior to experiments.
Morphometric analysis of acridine orange staining.
Cells were stained with 10 mM acridine orange in phenol red-free media for 1 h. Images of stained cells were acquired using fluorescence microscopy as described above. Images of 10 fields with 5 to 10 cells for each image were taken using multiple stage positions in Multidimensional Acquisition mode under conditions of no photobleaching (ND2 filter, 1000 msec exposure) that enabled the acquisition of multiple images of the same cells. Stage position for each image was stored so images of the same fields and cells could be taken before and after treatment. Phenol red-free media containing 10 mM acridine orange in the presence or absence of Meth or Clq was added to the cells. At the end of the incubation time, images of treated cells were taken and used for morphometric analysis. Change in acridine orange intensity was measured as a change in average pixel intensity in the cytoplasmic area using MetaMorph Version 6.1r6 image analysis software (Molecular Devices, Sunnyvale, CA). Background mean pixel intensity was measured in nuclear area and subtracted.
Ratiometric measurement of acidic organelle pH.
Dendritic cells were stained with 5 μM LysoSensor Yellow/Blue (Invitrogen, Carlsbad, CA) for 5 min before Meth (10, 50, 100 μM) or Clq (10μM) was added for an additional 10 min incubation followed by washing with phosphate-buffered saline (PBS) (pH 7.4). Fluorescent images were taken of the same cells using Olympus IX81 microscope with Photometrics CoolSNAP HQ cooled camera, MetaMorph Version 6.1r6 imaging software (Molecular Devices, Sunnyvale, CA), Olympus PlanApo 40x/1.4 Oil objective, equipped with fluorescent yellow customized Chroma (D350/50 excitation, 400DCLP dichroic splitter and D535/40m emission) (Chroma technology Corp., Rockingham, VT) and blue Chroma 31000v2 (D350/50 excitation, 400DCLP dichroic splitter and D460/50m emission) filter sets. Average pixel intensity was measured in the cytoplasm of the cells excluding the nucleus using MetaMorph software, and the ratio of yellow to blue intensity was compared to a pH calibration curve to determine pH values. For the calibration curve, cells were stained with 5 μM LysoSensor Yellow/Blue for 20 min, washed with PBS and incubated in buffer of known pH (4.0 to 7.4) containing 10 μM monensin and 10 μM nigericin [56] before images were taken and processed as above.
Ultrastructural analysis and immunogold labeling.
Dendritic cells were derived and incubated for 4 h with or without Clq (20 μM) or Meth (100 μM). Cells were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide followed by 1% uranyl acetate, dehydrated through a graded series of ethanol and embedded in LX112 resin (LADD Research Industries, Williston, VT). Ultrathin (80 nm) sections were cut on a Reichert Ultracut UCT, stained with uranyl acetate followed by lead citrate and viewed on a JEOL 1200EX transmission electron microscope at 80 kV.
For immunogold labeling, cells treated as described above were fixed in 2% paraformaldehyde and 4% polyvinylpyrolodone in phosphate buffer 0.2 M (pH 7.4) at 4 °C. Fixed cells were processed for ultrathin cryosectioning as previously described [14]. Immunogold labeling was performed using LAMP-1 antibody (clone 1D4B, BD Pharmingen, San Diego, CA) followed by anti rat Ig-G coupled with 10 nm gold particles and biotinylated anti-MHC II (clone M5/114.15.2, BD Pharmingen, San Diego, CA) followed by streptavidin gold (15 nm). Contrast was obtained with a mixture of 2% methylcellulose (Sigma, St. Louis, MO) and 0.4% uranyl acetate pH 4 (EMS, Hatfield, PA).
Antigen processing.
FITC-coupled BSA, ovalbumin or casein were fed to immature bone marrow-derived dendritic cells (between 1 to 3 × 107 cells for each condition) at a concentration of 100 μg/ml. Cells were untreated or treated with Meth or Clq. After overnight incubation, cells were washed twice in PBS and lysed in 150 mM NaCl, 50 mM Tris-HCl and 1% NP40 supplemented with protease inhibitor cocktail. Post-nuclear supernatants were normalized for protein content and 80 μg of total protein was run on SDS-PAGE gel. Membrane blots were probed with the anti FITC mAb or β-tubulin mAb (Sigma, St. Louis, MO).
Sub-cellular fractionation.
The immature bone marrow-derived dendritic cell line JAWS (ATCC) was used for sub-cellular fractionation. One hundred million cells were used for each experimental condition. Cells (4 × 106 cells/ml) were incubated overnight with 80 μg/ml of FITC-labeled BSA or casein in presence or absence of Clq (20 μM) or Meth (50–100 μM). Cells were then lysed in 250 mM sucrose, 1 mM EDTA pH 7.4. Early and late endosomes and lysosomes were prepared over consecutive Percoll gradients (27% and 10%) from cells treated as reported above [57]. Each fraction was tested for β−hexosaminidase to locate lysosomes and late endosomes. The late endocytic marker Lamp-1 (clone 1D4B, BD Pharmingen, San Diego, CA) and the early endosomes/plasma membrane marker transferrin receptor (TrfR) (clone M-A712, BD Pharmingen, San Diego, CA) were also used to assess the purity of the endosomal preparations. Pulled fractions 3–6 from the 27% Percoll gradient (lysosomes), 2–5 from the 10% Percoll gradient (late endosomes) and 7–10 from the 10% Percoll gradient (early endosomes) were run on SDS-PAGE and blotted membranes analyzed for FITC-labeled antigens as reported above.
Pulse chase and immunoprecipitation.
GM-CSF differentiated bone marrow dendritic cells were cultured in methionine- and cysteine-free medium complete DMEM media containing 5% dialyzed serum for 1 h. Cells were then labeled with 0.2 mCi/ml [35S]-methionine (Perkin Elmer, Waltham, MA) for 30 minutes (pulse). Cells were then washed three times and incubated in complete DMEM media supplemented with 10X cold methionine for 4 hours (chase) in the presence or absence of 50 μM Meth. Cells were subsequently lysed in 1% NP40, 150 mM NaCl, 50 mM Tris containing a cocktail of protease inhibitors (Complete Mini, Roche Diagnostics, Indianapolis, IN) for 30 min on ice, spun at 14000 rpm for 30 minutes to remove cell nuclei and debris. The amount of incorporated radioactivity in each sample was determined by precipitating 10 μl of the post-nuclear supernatants with 10% trichloroacetic acid (TCA). Equivalent amounts of radioactive lysates were pre-cleared with rat serum adsorbed to Prot G beads followed by Protein G beads alone, for 2 hours at 4°C. Immunoprecipitation was performed using 10 μg of anti CD74 (clone In-1, Pharmingen, San Diego, CA) bound to Protein G beads. The beads were washed 3 times with lysis buffer and eluted with sample buffer. The elute was boiled and resolved by SDS-PAGE. The gel was subsequently dried and exposed in autoradiography.
Antigen presentation.
Bone marrow-derived dendritic cells from OT II transgenic mice (Jackson Laboratory, Bar Harbor, MN) were grown in 10 ng/ml of mouse GM-CSF for 10–12 days. Splenic T cells were purified using the pan-T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's suggestions. One hundred thousand dendritic cells were cultured with 4 × 105 T cells in the presence or absence of 0, 3, 10, and 30 μM OVA protein for 3 days at 37°C. In some experiments Meth or Clq was added on the first day of culture until the end of the proliferative response. In other experiments, dendritic cells were pretreated with Meth or Clq for 4 h at 37°C, washed and fixed in 1% paraformaldehyde before adding OVA 323–339 peptide and the T cells. In all experiments, [3H]-thymidine (1 μCi/well) was added during the last 18 h of incubation to assay T cell proliferation. Plates were harvested and the DNA [3H]-thymidine incorporation was monitored using a Wallac liquid scintillation counter (Perkin Elmer, Waltham, MA).
Autophagy assay in GFP-LC3 dendritic cells and macrophages.
Bone marrow-derived dendritic cells and macrophages from GFP-LC3 animals were plated at 4 × 105 cells/cm2 density in glass bottom dishes and incubated overnight. Meth or Clq was added to the media, and the cells incubated for 2 h and 24 h before fluorescence microscopy using an Olympus IX81 microscope with Photometrics CoolSNAP HQ cooled camera, MetaMorph Version 6.1r6 imaging software (Molecular Devices, Sunnyvale, CA), TC-324B Automatic Temperature Controller (Warner Instrument Corporation, Hamden, CT), Olympus PlanApo 60x/1.4 Oil objective, and a Chroma FITC 41001 (HQ480/40x excitation, Q505LP dichroic splitter and HQ535/50m emission) (Olympus, Center Valley, PA) to determine GFP-LC3 puncta formation in the cells. At least 100 cell profiles per dish were assayed in triplicate for GFP-LC3 puncta.
Macrophage phagocytosis assay and colony forming unit (CFU) determination.
Mouse peritoneal macrophages at 3.5 × 105 cells/cm2 density on cover slips were incubated at 37°C with 20 μl of IgG opsinized sheep erythrocytes in 520 μl volume for 90 min. Uningested erythrocytes were lysed by sequential washing with PBS, water, and PBS. The phagocytic index was quantified by measuring the number of erythrocytes phagocytosed per 100 macrophages using bright field microscopy and 20x magnification, and inhibition is identified as percent of control. Data were collected from 4–7 independent experiments. The average control phagocytic index was 433.
To determine the phagocytic index for Candida albicans (Ca) and Cryptococcus neoformans (Cn) the macrophage like-cell line J774.16 (cultured in DMEM with 10% heat-inactivated FCS, 10% NCTC-109 medium, and 1% nonessential amino acids) was treated with 10 or 50 μM Methfor 2 h and then washed three times in media. As controls, J774.16 cells were incubated in medium alone or in the presence of 20 μM chloroquine. Ca strain SC5314 yeast cells were grown in brain heart infusion medium (BHI) at 37°C for 24 h then washed three times in PBS prior to application to macrophage. Cn yeast strain H99 was grown for 24 h in BHI at 37°C, incubated with capsule-specific monoclonal antibody (mAb) 18B7 [58] as an opsonin at 10 μg/ml for 2 h and then washed three times in PBS prior to incubation with macrophage. Ca and Cn cells were added to the macrophage monolayer at an effector to target ratio 1:1, and the suspension was incubated at 37°C for 30 min with Ca or 1 h with Cn. After incubation, remaining extracellular yeast cells were removed with three washes of PBS. The phagocytic index was determined by microscopic examination. For each experiment, five fields in each well were counted, and at least 100 macrophages were analyzed in each well. Wells were performed in triplicate for each condition examined.
Colony counts were made to determine the number of viable Ca and Cn yeast cells after phagocytosis. For the colony forming unit (CFU) determination, J774.16 macrophages were treated with or without Methor chloroquine and infected with Ca or Cn as described. The cultures were washed after 2 h to remove extracellular yeast and then incubated for an additional 22 h. After the 24 h total incubation, macrophage cells were lysed by forcibly pulling the culture through a 27-gauge needle 5 times. The lysates were serially diluted, and plated on Sabouraud dextrose agar at 37°C. CFU determinations were made after 72 h. Controls also consisted of yeast grown without macrophage, but in the presence of chloroquine or methamphetamine. All tests were preformed in triplicate.
Supporting Information
Figure S1. Meth Does Not Affect Cell Viability
Bone marrow-derived dendritic cells were treated with Meth (100 μM) or ethanol for 2 h at room temperature. Cells were then analyzed by flow cytometry for changes in cellular shape or granulosity both indicators of cell death. No differences in shape, morphology or granulosity were observed following Meth treatment.
Figure S2. Meth Does Not Affect Fluid Phase Phagocytosis
Bone marrow-derived dendritic cells pre-treated with Meth or Clq for 2 h or left untreated were incubated for 30 min with PE-conjugated 2 μm fluorescent dextran microbeads with a cell-bead ratio of 1:100. The efficiency of the uptake was quantified by flow cytometry. Numbers in each quadrant refer to percentage of cells that have internalized 0, 1, 2, and 3 or more beads (from left to right).
Figure S3. Meth Induces Rapid Alkalization in Acidic Organelles in Macrophages
(A) Representative fluorescence microscopy images of bone marrow-derived macrophages with reduced acridine orange staining following 10 min of Meth exposure Clq treatment. Scale bar, 10 μm. (B) Morphometric analysis of the decrease in acridine orange staining indicating alkalization upon 10 min treatment with Meth or Clq. Similar results were obtained in three independent experiments (n = 25–35, mean ± SEM, *p < 0.01, ***p < 0.0001, two-tailed ANOVA). (C) Progressive change in acridine orange label in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25–35, mean ± SEM, Student's two-tailed t-Test).
Figure S4. Meth Induces Autophagosome Accumulation in Macrophages as Lysosomal pH Is Alkalized
(A) Progressive accumulation of GFP-LC3 positive autophagosomes in Meth- and Clq-treated cells at 2 h and 24 h. (n = 300–400, mean ± SEM, **p < 0.001, ***p < 0.0001, Fisher's exact test.) (B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. (n = 300–400, mean ± SEM, ***p < 0.0001, Fisher's exact test.) (C) Fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with Meth or Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm. (D) Phagosomes and autophagosomes labeled by GFP-LC3, in untreated, Meth- or Clq-treated (2 h) mouse peritoneal macrophages phagocytosing sheep erythrocytes. Red arrows denote representative autophagosomes, while yellow arrows denote representative phagocytosed erythrocytes. Scale bar, 10 μm. (E) Morphometric analysis of monodansylcadaverine label in peritoneal macrophages after 24 h treatment with Meth (n = 30, mean ± SEM, ***p < 0.0001, two-tailed ANOVA).
Figure S5. P24 Production Is Increased in Meth-Treated JR-CSF/huCycT1 Monocytes
(A) Bone marrow dendritic cells from JR-CSF/huCycT1 mice were incubated with or without different concentration of Meth or NH4Cl. Supernatant was collected at day 7 and amount of secreted p24 measured. (Statistics n = 4 dishes, mean ± SEM, *p < 0.05, **p < 0.001, two-tailed ANOVA.) (B) JR-CSF/huCycT1 mice were left untreated or treated for one week with different amounts of Meth (low and high as described in materials and methods). Sera collected from each mouse were tested for amount of HIV-1 RNA using RT-PCR. The values are the mean and standard deviation from the values obtained from five mice in each group sample (n = 5 animals, mean ± SEM, two-tailed ANOVA).
Protocol S1. Supporting Methods
Table S1. Fluid-Phase Phagocytotic Uptake of Lucifer Yellow (LY) Is Unaffected by Meth and Clq Treatment in Mouse Peritoneal Macrophages
We thank Judith Rabkin (Columbia University) and Perry N. Halkitis (New York University) for information on patterns of recreational Meth use; Ana Maria Cuervo (Albert Einstein College of Medicine) for discussion of potential roles for autophagic pathways in antigen processing; Yvonne Schmitz, Kristine Larsen and Kim Atyeh for comments on the manuscript.
Author Contributions
The experiments were planned, performed, and analyzed jointly by ZT, JL, DS, and LS. JM, DJ, YR, AG, and ST performed the experiments. The GFP-LC3 mouse was constructed by NM. NM, JN, and HG contributed reagents/materials/analysis tools. DS and LS conceived and designed the experiments. The paper was written by ZT, JL, DS, and LS.
- 1.
Drug and Alcohol Services Information System (2006) Trends in methamphetamine/amphetamine admissions to treatment: 1993–2003. Available: http://www.oas.samhsa.gov/2k6/methTX/methTX.htm. Accessed 12 June 2007.
- 2. Colfax G, Shoptaw S (2005) The methamphetamine epidemic: implications for HIV prevention and treatment. Curr HIV/AIDS Rep 2: 194–199.
- 3. Urbina A, Jones K (2004) Crystal methamphetamine, its analogues, and HIV infection: medical and psychiatric aspects of a new epidemic. Clin Infect Dis 38: 890–894.
- 4. Ellis RJ, Childers ME, Cherner M, Lazzaretto D, Letendre S, et al. (2003) Increased human immunodeficiency virus loads in active methamphetamine users are explained by reduced effectiveness of antiretroviral therapy. J Infect Dis 188: 1820–1826.
- 5. Carey CL, Woods SP, Rippeth JD, Gonzalez R, Heaton RK, et al. (2006) Additive deleterious effects of methamphetamine dependence and immunosuppression on neuropsychological functioning in HIV infection. AIDS Behav 10: 185–190.
- 6. Cherner M, Letendre S, Heaton RK, Durelle J, Marquie-Beck J, et al. (2005) Hepatitis C augments cognitive deficits associated with HIV infection and methamphetamine. Neurology 64: 1343–1347.
- 7. Gonzales R, Marinelli-Casey P, Shoptaw S, Ang A, Rawson RA (2006) Hepatitis C virus infection among methamphetamine-dependent individuals in outpatient treatment. J Subst Abuse Treat 31: 195–202.
- 8. (No Authors Listed) (2005) Meth's impact on HIV epidemic being studied. Drug's use is growing problem among MSMs. AIDS Alert 20: 79–80.
- 9. Torian LV, Blank S, Kellerman SE, Frieden TR, Ho DD, et al. (2006) Investigation of a new diagnosis of multidrug-resistant, dual-tropic HIV-1 infection–New York City, 2005. MMWR Morb Mortal Wkly Rep 55: 793–796.
- 10. Yu Q, Zhang D, Walston M, Zhang J, Liu Y, et al. (2002) Chronic methamphetamine exposure alters immune function in normal and retrovirus-infected mice. Int Immunopharmacol 2: 951–962.
- 11. In SW, Son EW, Rhee DK, Pyo S (2005) Methamphetamine administration produces immunomodulation in mice. J Toxicol Environ Health A 68: 2133–2145.
- 12. Theodore S, Cass WA, Maragos WF (2006) Involvement of cytokines in human immunodeficiency virus-1 protein Tat and methamphetamine interactions in the striatum. Exp Neurol 199: 490–498.
- 13. Mahajan SD, Hu Z, Reynolds JL, Aalinkeel R, Schwartz SA, et al. (2006) Methamphetamine modulates gene expression patterns in monocyte derived mature dendritic cells: implications for HIV-1 pathogenesis. Mol Diagn Ther 10: 257–269.
- 14. Semple SJ, Patterson TL, Grant I (2003) Binge use of methamphetamine among HIV-positive men who have sex with men: pilot data and HIV prevention implications. AIDS Educ Prev 15: 133–147.
- 15. Chang L, Ernst T, Speck O, Grob CS (2005) Additive effects of HIV and chronic methamphetamine use on brain metabolite abnormalities. Am J Psychiatry 162: 361–369.
- 16. Riviere GJ, Gentry WB, Owens SM (2000) Disposition of methamphetamine and its metabolite amphetamine in brain and other tissues in rats after intravenous administration. J Pharmacol Exp Ther 292: 1042–1047.
- 17. Kleijmeer MJ, Raposo G, Geuze HJ (1996) Characterization of MHC class II compartments by immunoelectron microscopy. Methods 10: 191–207.
- 18. Paludan C, Schmid D, Landthaler M, Vockerodt M, Kube D, et al. (2005) Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 307: 593–596.
- 19. Lee HK, Lund JM, Ramanathan B, Mizushima N, Iwasaki A (2007) Autophagy-dependent viral recognition by plasmacytoid dendritic cells. Science 315: 1398–1401.
- 20. Talloczy Z, Virgin HW 4th, Levine B (2006) PKR-dependent autophagic degradation of herpes simplex virus type 1. Autophagy 2: 24–29.
- 21. Talloczy Z, Jiang W, Virgin HW 4th, Leib DA, Scheuner D, et al. (2002) Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc Natl Acad Sci U S A 99: 190–195.
- 22. Melega WP, Cho AK, Harvey D, Lacan G (2007) Methamphetamine blood concentrations in human abusers: application to pharmacokinetic modeling. Synapse 61: 216–220.
- 23. Cho AK, Melega WP, Kuczenski R, Segal DS (2001) Relevance of pharmacokinetic parameters in animal models of methamphetamine abuse. Synapse 39: 161–166.
- 24. Harris DS, Boxenbaum H, Everhart ET, Sequeira G, Mendelson JE, et al. (2003) The bioavailability of intranasal and smoked methamphetamine. Clin Pharmacol Ther 74: 475–486.
- 25. Wilson JM, Kalasinsky KS, Levey AI, Bergeron C, Reiber G, et al. (1996) Striatal dopamine nerve terminal markers in human, chronic methamphetamine users. Nat Med 2: 699–703.
- 26. Karch SB, Stephens BG (2000) Toxicology and pathology of deaths related to methadone: retrospective review. West J Med 172: 11–14.
- 27. Logan BK, Fligner CL, Haddix T (1998) Cause and manner of death in fatalities involving methamphetamine. J Forensic Sci 43: 28–34.
- 28. Trombetta ES, Mellman I (2005) Cell biology of antigen processing in vitro and in vivo. Annu Rev Immunol 23: 975–1028.
- 29. Riese RJ, Mitchell RN, Villadangos JA, Shi GP, Palmer JT, et al. (1998) Cathepsin S activity regulates antigen presentation and immunity. J Clin Invest 101: 2351–2363.
- 30. Eskelinen EL, Illert AL, Tanaka Y, Schwarzmann G, Blanz J, et al. (2002) Role of LAMP-2 in lysosome biogenesis and autophagy. Mol Biol Cell 13: 3355–3368.
- 31. Peters PJ, Neefjes JJ, Oorschot V, Ploegh HL, Geuze HJ (1991) Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature 349: 669–676.
- 32. Nishi T, Forgac M (2002) The vacuolar (H+)-ATPases–nature's most versatile proton pumps. Nat Rev Mol Cell Biol 3: 94–103.
- 33. Hurtado-Lorenzo A, Skinner M, El Annan J, Futai M, Sun-Wada GH, et al. (2006) V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat Cell Biol 8: 124–136.
- 34. Sulzer D, Maidment NT, Rayport S (1993) Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons. J Neurochem 60: 527–535.
- 35. Sulzer D, Sonders MS, Poulsen NW, Galli A (2005) Mechanisms of neurotransmitter release by amphetamines: a review. Prog Neurobiol 75: 406–433.
- 36. Zwart W, Griekspoor A, Kuijl C, Marsman M, van Rheenen J, et al. (2005) Spatial separation of HLA-DM/HLA-DR interactions within MIIC and phagosome-induced immune escape. Immunity 22: 221–233.
- 37. Carballo E, Pitterle DM, Stumpo DJ, Sperling RT, Blackshear PJ (1999) Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts. Am J Physiol 277: C163–C173.
- 38. Barnden MJ, Allison J, Heath WR, Carbone FR (1998) Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol Cell Biol 76: 34–40.
- 39. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15: 1101–1111.
- 40. Larsen KE, Fon EA, Hastings TG, Edwards RH, Sulzer D (2002) Methamphetamine-induced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis. J Neurosci 22: 8951–8960.
- 41. Richards JR, Bretz SW, Johnson EB, Turnipseed SD, Brofeldt BT, et al. (1999) Methamphetamine abuse and emergency department utilization. West J Med 170: 198–202.
- 42. Bouvier G, Benoliel AM, Foa C, Bongrand P (1994) Relationship between phagosome acidification, phagosome-lysosome fusion, and mechanism of particle ingestion. J Leukoc Biol 55: 729–734.
- 43. Cox D, Lee DJ, Dale BM, Calafat J, Greenberg S (2000) A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis. Proc Natl Acad Sci U S A 97: 680–685.
- 44. Enoch DA, Ludlam HA, Brown NM (2006) Invasive fungal infections: a review of epidemiology and management options. J Med Microbiol 55: 809–818.
- 45. Sun J, Soos T, Kewalramani VN, Osiecki K, Zheng JH, et al. (2006) CD4-specific transgenic expression of human cyclin T1 markedly increases human immunodeficiency virus type 1 (HIV-1) production by CD4+ T lymphocytes and myeloid cells in mice transgenic for a provirus encoding a monocyte-tropic HIV-1 isolate. J Virol 80: 1850–1862.
- 46. Savarino A, Di Trani L, Donatelli I, Cauda R, Cassone A (2006) New insights into the antiviral effects of chloroquine. Lancet Infect Dis 6: 67–69.
- 47. Zhou D, Li P, Lin Y, Lott JM, Hislop AD, et al. (2005) Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens. Immunity 22: 571–581.
- 48. Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, et al. (2005) Autophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc Natl Acad Sci U S A 102: 7922–7927.
- 49. Levitz SM, Harrison TS, Tabuni A, Liu X (1997) Chloroquine induces human mononuclear phagocytes to inhibit and kill Cryptococcus neoformans by a mechanism independent of iron deprivation. J Clin Invest 100: 1640–1646.
- 50. Harrison TS, Griffin GE, Levitz SM (2000) Conditional lethality of the diprotic weak bases chloroquine and quinacrine against Cryptococcus neoformans. J Infect Dis 182: 283–289.
- 51. Hart PD, Young MR (1991) Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast. J Exp Med 174: 881–889.
- 52. Raposo G, Moore M, Innes D, Leijendekker R, Leigh-Brown A, et al. (2002) Human macrophages accumulate HIV-1 particles in MHC II compartments. Traffic 3: 718–729.
- 53. Deneka M, Pelchen-Matthews A, Byland R, Ruiz-Mateos E, Marsh M (2007) In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53. J Cell Biol 177: 329–341.
- 54.
The Partnership for a Drug-Free America (2007) Partnership for a Drug-Free America. Available: http://www.drugfree.org/. Accessed 20 September 2007.
- 55. Yin B, Loike JD, Kako Y, Weinstock PH, Breslow JL, et al. (1997) Lipoprotein lipase regulates Fc receptor-mediated phagocytosis by macrophages maintained in glucose-deficient medium. J Clin Invest 100: 649–657.
- 56. Diwu Z, Chen CS, Zhang C, Klaubert DH, Haugland RP (1999) A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells. Chem Biol 6: 411–418.
- 57. Castellino F, Germain RN (1995) Extensive trafficking of MHC class II-invariant chain complexes in the endocytic pathway and appearance of peptide-loaded class II in multiple compartments. Immunity 2: 73–88.
- 58. Casadevall A, Cassone A, Bistoni F, Cutler JE, Magliani W, et al. (1998) Antibody and/or cell-mediated immunity, protective mechanisms in fungal disease: an ongoing dilemma or an unnecessary dispute. Med Mycol 36(Suppl 1): 95–105.


text:
"My experience at the FAO meant much more for me than what I had expected. It enriched my values, increased and enhanced my knowledge and skills, and helped me grow each day both personally and professionally. I had the honour of working with the top professionals in the fields in which I would like to build a career and to do so in a multicultural setting that was much more interesting and enriching than I could have imagined."
Made his internship at the UN Food and Agriculture Organization (FAO)
Ex Student of the Master in Diplomacy and Public International Service (2011-2012)

subset: commoncrawl


text:
Cities in northwest Broward are attempting to make this Halloween a memorable one for people in the community by organizing several spooky festivities, suitable for children, as well as the child within the adult.
Coral Springs' Halloween Haunted House will take place on Oct. 23, 5 to 8 p.m., and on Oct. 24, 2 to 7 p.m., at the Coral Springs Gymnasium, 2501 Coral Springs Drive, in Coral Springs. Participants will be able to wander through a non-scary haunted house and play on bounce houses and slides. The cost is $5 per child and $2 per adult. Additional information can be obtained by calling Parks and Recreation at 954-345-2200.
Halloween Spooktakular with Grizelda the Witch, for children age 3 to 7, will take place on Oct. 26, 4 to 5 p.m., at the Parkland Library, 6620 University Drive, in Parkland. Story-telling, magic and treats will be a part of the event. Children older than 8 years old will be able to enjoy "Ghostly Gathering" on Oct. 27, 6 to 7 p.m., at the library.
A Parkland Library card is required to register for both of the events. Register by calling 954-757-4207 or visiting CityOfParkland.org/library.
Parkland's third annual Halloween festival, Trunk or Treat, will be Oct. 29, 4 to 7 p.m., at the Equestrian Center at Temple Park. Participants can come dressed in costume and stroll through the Trunk or Treat Lane, pick out pumpkins from Parkland's Pumpkin Patch, and participate in family arts and crafts. Children will "trick or treat" for candy out of decorated car or trunk beds. The cost is $20. Applications to participate must be filled and mailed to City of Parkland, Attention: Gayle Vasile, 10561 Trails End, Parkland, Fla., 33076, before Oct. 22.
Latest Coral Springs News
The Coconut Creek Community Center is also presenting two Halloween events. Children, age 3 to 6, can celebrate early during Halloween Spooktacular on Oct. 23, 6 to 7:30 p.m. Parental participation is mandatory. The cost is $15 for Coconut Creek residents and $20 for non-residents, and includes the classic "It's the Great Pumpkin Charlie Brown" movie.
Children can also paint a pumpkin and a trick-or-treat bag in two sessions: Oct. 19, 6 to 7 p.m., for age 5 to 10, and on Oct. 30, 10 to 11 a.m., for children age 2 to 4. The cost is $10 for residents and $15 for non-residents. The Community Center is located at 1100 Lyons Road in Coconut Creek.
The Howl-O-Ween Dog Costume Contest will take place Oct. 26, 6 to 7 p.m., at the Dog Park in Windmill Park, 700 Lyons Road, Coconut Creek. Dogs dressed in costume will receive a goody bag. Prized will be awarded. Admission is free. Registration is through Oct. 16.
Those who are interested can register at CoconutCreek.net/webtrac or by walk-in at the Community Center. Call 954-545-6670.


text:
2015 Chevrolet Suburban Values
Select a 2015 Chevrolet Suburban trim level
2015 model year changes
Chevrolet's full-size Suburban and Tahoe SUVs, have been completely redesigned for 2015. While these models are essentially the same size on the outside as those that they replace, GM has re-engineered these trucks for much-improved refinement and ride quality--as well as more power and higher towing capability. Both model lines get new Chevrolet MyLink touch-screen connectivity in most of their trim levels. Meanwhile, second- and third-row seating has been revamped to include the convenience of a fold-flat arrangement, for added cargo versatility. Additionally, Suburban models get a little more legroom for those in the second row, while a new dual-screen rear entertainment system with Blu-ray DVD player is offered in the Suburban. Otherwise keyless entry and push-button start are both new and widely available in both the Suburban and Tahoe lineups, as are other new convenience features, like adaptive cruise control.
Competitive comparison
Much-improved interior; seating space; versatility and toughness; towing ability; comfortable, refined ride; relatively fuel-efficient engines
Special features for 2015
The Suburban and Tahoe are by far the best-selling models in their class, and they're tough rivals for the Ford Expedition, Toyota Sequoia and Nissan Armada, among just a few others. These two Chevy models are closely related in most ways--although there's a key difference: The Suburban is 14 inches longer in wheelbase and 20 inches longer overall than the Tahoe; and that results in a significantly larger third-row seat than in the Tahoe--as well as easier access to it, and more cargo space. Compared to rival models, the Chevy Tahoe and Suburban still stand out for their good ride quality and efficient engines, and they handle surprisingly well considering their size and mass. They're also very rugged and versatile, and can be outfitted for off-road toughness, tow ability, and interior luxury as needed. Chevrolet claims that the Suburban will be the most fuel-efficient model in the full-size SUV class, thanks to its standout combination of fuel-saving technologies like direct injection and active fuel management (cylinder deactivation). Although the profile of the Suburban and Tahoe haven't changed all that much, Chevy has managed to make significant improvements to aerodynamics. Yet chiseled sheetmetal and big, flamboyant wheels (up to 22 inches) help maintain a powerful look.


text:
Speaking at the Forbes 30 under 30 summit, former White House intern Monica Lewinsky explained to the audience that she was "patient zero" for being bullied on the internet.
.@MonicaLewinsky joins Twitter, tells Forbes Under 30 crowd in Philadelphia, "My life was ruined by @DRUDGE" pic.twitter.com/BD4Sv58I0J
— James O'Keefe (@JamesOKeefeIII) October 20, 2014
Lewinsky: "It was like a punch in the gut… Thankfully, people aren't punched every day on the street but it happens all the time online."
— Stephanie Farr (@FarFarrAway) October 20, 2014
"I lost my public self, or had it stolen… In a way, it was a form of identity theft." –@monicalewinsky #Under30Summit
— Jeff Bercovici (@jeffbercovici) October 20, 2014
"There are those who say to me, 'Monica, why don't you shut up?' They will say it today. They never shut up." –@monicalewinky #under30summit
— Jeff Bercovici (@jeffbercovici) October 20, 2014


text:
Hockey India drops its High Performance Director David John from selection panel
New Delhi, Oct 2 (PTI) Hockey India (HI) Tuesday decided to drop its High Performance Director David John from the men's senior team selection committee for showing "bias" against a few senior players.
Expressing his displeasure at John discussing with the media India's disappointing performance in the Asian Games and its likely fallouts on some players, newly-elected HI President Mushtaque Ahmad decided to remove him from the selection panel.
In a letter to his Secretary General Rajinder Singh, the HI chief said John will not be a part of the selection committee which will pick the Indian team for the upcoming World Cup in November-December. But sources said that John will not be a part of any selection panel from now on.
"I am not happy with David John commenting on individual players who played in Asian Games. As a matter of policy/principle we do not allow discussion on individual players in public domain and John by discussing that has broken all protocols," Mushtaque, who was elected as HI chief on Monday, said.
"Observing the bias of David John against few players of senior men's team, I have decided that John will not be a part of the senior men's selection committee for the World Cup. This is to ensure fairness in selection without any prior bias against anyone in anyone's mind," he said.
Mushtaque also asked Rajinder to report to him after discussing with John as to what he has done in the last two years for the development of Indian hockey at grass root level, on the identification of academies and development of coaching programme for Indian coaches.
A top HI source, however, said that "as of now" John's contract which runs till 2020 Tokyo Olympics is safe.
"As of now, John will continue as High Performance Director. There is no change in that, but he will not henceforth be a part of any selection committee of HI," a source from the HI told PTI on condition of anonymity.
John left for Argentina on Tuesday as part of Indian contingent for the Youth Olympic Games in Buenos Aires. India is fielding both men's and women's teams in Hockey 5s competition in the Youth Olympics


text:
The Three Laws of User Interface Design
When it comes to user interface design, there are things on the user's side that need to be considered. The goal of any user interface is to make it easier and more enjoyable for users to work with, so it's important to always try and improve your customer experience. As a designer you should always keep the following three laws in mind when creating a new user interface or editing an existing one.
The Law of Clarity
Always remember Google and their lack of following the Law of Clarity in terms of their latest Gmail update. This law states that your user will avoid elements in the interface without a clear meaning. During a previous Google update, they moved all connected programs from Gmail, such as Calendar, Drive, Youtube and the Play Store behind an ambiguous Grid box icon. The reason that this example is important is that Google got flooded with email requests wanting to know where the other applications went. In general, since your customers are in the "Want it now, buy it now" mindset, if they cannot immediately understand the something, they will ignore it, never bothering to find out.
The Law of Defaults
The second law to keep in mind in user interface design is the Law of Defaults. This law means that your user will rarely change the default settings. Most notably is the most popular ringtone on the planet – the Nokia default ring tone. Most people never bother to change the default settings, such as their background and ringtones on their phones, nor the default fridge setting for the temperature. Defaults can be very powerful. To this end, make sure your default settings are as practical and useful as possible.
The Law of Guided Action
The third important law is the Law of Guided Action. Your user will more than likely do something if they are asked to do it. There is a large difference between asking a user to do something and expecting them to know to do it. Case in point, LinkedIn introduced its Endorsements feature and took the initiative to create highly-visible, call-to-action banners that appeared at the top of the profile pages.
To create a user interface that is both interactive and provides for the laws of clarity, defaults and guided actions, you must create an interface that makes use of the dialogue of the user, while assuming that both experienced and new users are seeing your work for the first time. Never assume all users will understand all the time and make it as easy as possible for them to use your interface.
The Bold Circle specializes in user interface design and our experienced team can help you to improve your current UI or create a brand new one for your customers.
Connect with us today to find out more about our user interface design solutions.


text:
Freaking News
Funny Body Pictures
My Country, My Body, My Choice..
Funny Two Chips in The Great North Korean body
Kim has two Chips. McDonald's chip in his stomach and nuclear chip below.
Funny Fireman's body for next movie
Member reactions:
Especially this pink nose. Congratulations Looks figurative
Funny Leonardo's body fans from Democratic Party
Funny Body Replacement
Funny Removal parasites from candidate's body
Scientists like Barack Obama so much that they named a parasite after him
Member reactions:
Great chop and you for sure win for best news story. Cracks me up.
Funny Camel with an Upside Down Body
Member reactions:
Great Work but, I ain't riding that thing home.
Funny Vladimir Putin the Body Builder
Member reactions:
Much better with some reflections on is head.
Funny Barack Obama as a Female Body Builder
Funny Gymnast Body Slamming a Wrestler
Member reactions:
I like the blending too, but she looks bigger than man.
i think it looks fine...she's falling on top of them... the rest glad you liked my design
with the votes im not surprised...it is whst is....i know improving a lot
Funny Body Scanners
England is implementing full body scanners in their airports. When conventional metal detectors trigger an alarm, the selected passengers must pose inside the scanner, which sees through their clothing to detect contraband underneath. The rule is simple - "if passengers refuse, they don't fly". There has been much controversy in the US about whether to implement such scanner machines, for ethical reasons. Many passengers don't feel comfortable being seen "without clothes" by airport security people. Use any types of body scanners to show what some celebrities may be hiding under their clothes. Make sure your entries are Safe For Work. Airport body scanners, portable body scanners, body scanner glasses, any body scanner gadgets can be used in this contest. Here's a good example by Franko.
Funny Body Parts
With the recent health care advances, more and more people receive complex organ transplants which were not possible years ago. The trend even got into Hollywood: the recent movie "The Eye" (2008) shows the main heroine undergoing a cornea transplant which gives her strange visions. The 2003 Oscar-winning movie "21 Gram" shows a person who receives a heart transplant, and how it changes his life. Photoshop how life would be different if the human body were easily assembled with the replaceable body parts (just like cars or any other industrial products). Here is a good example. You are encouraged to be more creative than just doing cyborgs.
Funny Out-of-body Experience
For the first time in history scientists were able to recreate out-of-body experiences without any use of drugs, in as many as 10 participants of the scientific experiment. The ten people reported seeing their own bodies from another location while being absolutely awake. This sensation caused some participants to get scared, but others were simply excited. This experiment is considered an important step to broadening the understanding of relationship between consciousness and human body. Photoshop out-of-body experience by showing ghost-like human soul next to its live body. Choose celebrities, politicians and any famous characters, and show how soul may disagree (or agree) with what a body is doing. Examples may be Lindsay Lohan trying to get in the car drunk, while her soul is trying to stop her. You can change the size and shape of a soul, but try to make it recognizable (e.g. soul of Donald Trump should at least resemble him somehow) and make sure both soul and body of the same person are in the picture.
Funny Body Jobs
Replace any body part with mechanical or electrical parts.
Funny Extra Body Parts
Create new and odd appendages for humans including new hands, new feet, legs and toes.
For more funny photoshop pix visit
Freaking News front page


text:
Darts Tournament I Every Sunday I Vancouver Wa.
Every Sunday we host Darts and Drafts. Darts and Drafts is a dart tournament where anyone (21+) can join in the fun. It is free to play and be a part of the tournament. Sign up starts at 5 pm. Dart games start at 5:30 p.m. The tournament is single elimination, and the winner wins a $15 gift card and all the glory for the week!
Join us for some darts, some craft beer and meet other dart enthusiasts!


text:
Midwifery in Texas & You
About midwives in Texas
There are two types of midwives which may legally practice in the state of Texas: Certified Nurse Midwives (CNM) and Licensed Midwives (LM). They may provide care in homes, birth centers, or hospitals.
Midwife means "with woman" and is a woman-centered, empowering model of maternity care. Midwives are trained professionals who focus on supporting women during pregnancy, birth, and the postpartum period.
The midwifery model of care respects pregnancy and birth as a normal, although life-changing, event. The midwifery model of care monitors the physical, emotional, spiritual and social well-being of the mother throughout the childbearing cycle, uses life-saving technological interventions only when truly needed and provides the family with individualized education, counseling, and prenatal care. Every midwife must maintain current certifications in First Aid, CPR and Neonatal Resuscitation as well as participating in continuing education and peer review yearly.
To read more about the the different types of midwives and the care they provide, see the links provided here.
How you can help
While many families in Texas have been cared for by licensed midwives for years, it hasn't always been an easy road. At times midwifery, home birth, birth centers, and your right to choose those things have come under fire in the Texas Legislature. We believe that all families should have the freedom to select the setting and provider that best fits their healthcare needs - whether that be at home, in a birth center, or in the hospital. To continue to preserve those rights takes hard work - and we need YOUR voice!
We have compiled a list of ways that you can help promote, encourage, and increase access to midwifery care for all women in Texas.
- Leave a review for your midwife on her business website and social media platforms.
- Share your birth experience with friends and family.
- Join consumer groups that support the midwifery model of care.
- Contact your elected officials and tell them about your experience with midwives.


text:
Underwater Whimsy
Underwater Whimsy
Honu (turtles) and Dolphins are both beloved creatures of the sea. They live in abundance in the waters off the Hawaiian Islands. While I've never seen them play together, it brought joy to my heart to think of them as playing games with balls and bubbles in the ocean.
Play is an action that benefits all of us, when we remember to take the time to play! We tend to take ourselves, our lives, our tasks too seriously. It's not that we shouldn't take our lives, our tasks, or ourselves seriously; it's more that we need to do so with a grain of salt and a hefty dose of balance.
Life is precious and meaningful, not to be wasted or spent worrying about what might happen or what has happened. We each have one day at a time in which to live. Let's do so to our fullest ability, remembering that FUN and PLAY are part of a happy, well-balanced life.
May these playful creatures tickle your fancy and remind you to PLAY!
Original Watercolor:
11"Hx15"W: $795 unframed
Open-edition Paper Giclée prints:
8"Hx10"W (image is 5"Hx6.75"W) = $40
11"Hx14"W mat (image is 7.5"Hx10.5"W) = $55
16"Hx20"W mat (image is 10.5"Hx14.5"W) = $95
Limited-edition, Gallery-wrapped, Canvas Giclée prints:
Image size: 16"Hx20"W gallery-wrapped, stretched canvas = $400.00
Image size: 16"Hx20"W gallery-wrapped, unstretched canvas, ready-to-assemble (stretcher bars not included) = $275.00
Image Size: 18"Hx24"W gallery-wrapped, stretched canvas = $500.00
Image Size: 18"Hx24"W gallery-wrapped, unstretched canvas, ready-to-assemble (stretcher bars not included) = $350.00
Image Size: 24"Hx36"W gallery-wrapped, stretched canvas = $750.00
Image Size: 24"Hx36"W gallery-wrapped, unstretched canvas, ready-to-assemble (stretcher bars not included) = $650.00
Shipping: All prices include standard shipping within the United States. For international shipping please contact Patrice.


text:
Become a part of Tu Nidito's future by partnering with us to support the Tu Nidito Endowment fund. The endowment fund is a fund of restricted money that will allow Tu Nidito ongoing support. The fund is structured so that the principal is kept in perpetuity and the annual disbursement becomes income for the agency. So what does this really mean? Right now, Tu Nidito begins every year with our budget at a zero balance. We work hard each year to raise the approximately million dollars it takes to ensure our support services are available to our community's children at no cost to them. An endowment will start our budget year with a positive. And we would love for you to be a part of it!
Tu Nidito's Federal Identification Number is: 86-0769031
Legal Name and Address:
Tu Nidito Children and Family Services
3922 N Mountain Avenue
Tucson, AZ 85719
Lisa Robinson, Director of Development
520-322-9155, ext 7013
Tu Nidito partners with the Jewish Community Foundation to manage the investment of endowment funds. Please contact the JCF, 520-577-0388, or Tu Nidito for more information or questions regarding making a donation or planned gift to the Tu Nidito Endowment.
Tu Nidito Founding Partners
A special thank you to Tu Nidito's Founding Endowment Partners who are helping to lead us to our vision where no child grieves alone. Thanks to the generosity of an anonymous donor, all founding gifts were matched up to $50,000.


text:
As most of you know. My wife Inga "Kitty" Trimm spent almost all of August and September hospitalized, much of it in ICU on a ventilator. (The picture was her in ICU) She had three major surgeries in that time. I honestly did not know if I would bring her home alive. But now we have been home from the hospital a few days. She is still attached to a "wound vac" by about about an eight foot tube, and can only get around with a walker, but she is home, and for that I am grateful.
However, over 50 days in the hospital wiped us out! This left us with many bills are past due, not to mention medical bills and we just don't have it. We absolutely must raise about $1,000. By the end of the day Friday ($2OO of that is needed today).
My wife Kitty wanted to add this:
This has also been difficult as to the fact I need his help a lot. I cannot cook, clean, bath, wash my hair, walk alone at all. I am constantly needing assistance by him. He cannot be gone long from home because I need his help just to go to the restroom. Please pray for him and please help if you can. He has a heavy burden and is trying to keep an upbeat attitude. Though I know it drains on him at times. I have a nurse that attends to me 3 times a week at home. That's how much help I am still needing. Thank you for all your help. Asking for the continued help and support.
If you can help, donations can be sent by PayPal to firstname.lastname@example.com
Views: 59
© 2019 Created by James Trimm. Powered by


text:
AIT SLIDE TALK #31 Gina Buenfeld
Talk by Gina Buenfeld from the Camden Arts Centre
Date and Time: Tuesday, September 9th, 19:00-21:00
Venue: AIT Room, Daikanyama
*The talk will be held in English, with no Japanese translation.
We are pleased to be hosting Gina Buenfeld, the Exhibition Organizer at the Camden Arts Centre for a one month residency in Tokyo supported by Cultural Affairs. (The residency will take place over a course of two periods, September 2014 and February 2015)
Gina Buenfeld will introduce the history of this institution, that has been one of London's leading publicly funded spaces for international contemporary art for the past 25 years. The talk will cover the Centre's activities which range from ambitious exhibitions with artists of the highest calibre from across the international arts' scene as well as an integrated public programme of talks, events, performances and screenings and a pioneering education programme. Camden Arts Centre has a distinctive combination of galleries, studios and workshop spaces and operates a highly regarded residency scheme. The discussion will place Camden Arts Centre within the broader London and UK art scene and will introduce other key spaces, educational facilities and artists based in the country as well as giving an overview of the current financial conditions and challenges for cultural production in the UK.
Throughout the talk, questions and comments from the audience will be welcomed.
We look forward to your attendance at this special event.
Date and Time: Tuesday September, 9th 19:00-21:00
Venue: AIT Room, Daikanyama (Twin Bldg. Daikanyama B-403, 30-8 Sarugaku-cho, Shibuya-ku, Tokyo) [MAP]
Organized by Arts Initiative Tokyo [AIT]
*The Agency for Cultural Affairs, Government of Japan, in Fiscal Year 2014
Booking required (limited to 40 guests)
Admission: JPY1000 (JPY800 for Students and AIT Base Members / Free for AIT House and Support Members)
Other details: Admission includes one free drink *Talk will be in English only.
Send an email with its subject as "AIT SLIDE TALK #31" to firstname.lastname@example.com, including your name, and contact.
[Speaker Biography]
Gina Buenfeld
2014-9- 1


text:
Tuesday, January 11, 2011
Canister Fun
8 comments:
They do look awesome! I just did mine today!! I was over at Thrify Decor Chick and saw yours...too funny! We did the same type of project a day apart! Angie xoReplyDelete
They turned out great!ReplyDelete
How sweet and I love them!ReplyDelete
How sweet of him to give you that HL gift card and you did a great job on finding these CUTE jars!ReplyDelete
Thanks for sharing your canisters at A Crafty Soiree! Are those stickers/rub ons you used to cuten them up? I love Hobby Lobby, your husband is sweet and knows you well. That was nice of him to surprise you. Please join us next time too!ReplyDelete
I love it. I hear on not finding anything when you have money or a gift card. I can find everything when I can't have it.ReplyDelete
Hi there! I'm your newest follower. I found you on the Friday link up. Lovely blog:) You can find me at www.bouffeebambini.blogspot.comReplyDelete
This is adorable and so is your family
Super cute! I found you from your link on Remodelaholic!ReplyDelete


text:
Online Collectibles Auctions
create account
sign in
browse auctions
search items
your account
Auction Network
Annual Labour Day Blowout Sale! - Three Sessions
Annual Labour Day Blowout Sale! - Three Sessions
Auction Currency
Auction Location
501 Alden Road
Auction Details
Our family of professional auctioneers, Allen, Ryan and Zach are pleased to bring you our Annual Labor Day Weekend Blowout Sale. This sale will include three live online sessions: September 2nd, 2017, September 3rd, 2017 and Labour Day Monday, September 4th, 2017. Auction Network is honoured to bring you a TRUE live online auction experience.
You will not want to miss this special three session sale!
Royal Canadian Mint Coins Including: 10oz Fine Gold Confederation Medals: 1867, 1927, 2017 (Issue Price $26,995 Each), 1806 USA 25c, RARE 1795 US $1000 Treasury Bond Unredeemed (Estimate $25,000), Australia 2000 1oz Gold World Trade Center Recovered Coin, Banknote Collection, Original Federal Reserve Note Bundles of 100, ICCS Collection of Canadian Coins, Over 600 Royal Canadian Mint Coins, Gold, Silver and Platinum Bullion, Private Collection of Original Oil Paintings: Christine Marshall, John Flaxman, Norval and Christian Morrisseau, Rare Canoe Painted by Norval Morrisseau, Estate Furniture From Gallery: Ethan Allen, Kaufman of Collingwood, Accent Tables, Drop Front Secretary Desk, Duncan Phyfe End Tables, Leather Wing Chair, 72x38" Executive Desk, 66x21" Hardwood Desk, Coffee and End Tables, Sports Memorabilia Collection of Signed Jerseys, Collector Frames, Pucks, NHL, NFL, MLB, Royal Doulton Figurines, Designer to the Stars Jewellery Collection Appraised and Certified (One of a Kind Statement Pieces, All Handmade in USA and Canada), Comic Book Collection, Rare Magazines & Newspapers, Antique Dolls, Die-cast Collection of Corgi, Lledo, Matchbox and Dinky, Jewellery Inventory of Loose Gemstones, Large Single Stones and Parcel Lots, Ladies 18kt Gold Cartier Panther Watch, Collection of Estate Sterling Silver Pieces and Much More!
Check back for special updates regarding this three session sale.
Debit, Cash, Visa, MasterCard, E-Transfer or Wire Transfer.
18% Buyers Premium, 2% Discount on E-Transfer payments.
Invoices will be charged to the credit card posted on registration following the auction sale. We must be notified by email (email@example.com) prior to the sale conclusion if you plan to pay by Debit, Cash, E-Transfer or Wire Transfer.
Choose Your Layout >
Auction Items Coming Soon!
There are no items currently viewable for this auction,
but they will be available here as soon as they are ready.
Auction Concluded
The auction has finished, bidding is closed.
If you won any items an email will be sent to you with invoice & payment information.
603 Items in this Session
Auctioneer Phone: 905-474-9743
View Preview Instructions
View Premiums/Taxes/Fees
View Payment & Shipping Info
View Terms & Conditions
Printable Items Listing
Ask Auctioneer a Question...
Auction Network
Markham, Ontario, Canada
About Auctioneer...
Auctioneer's Auctions...
Recently Viewed Items
About iCollector.com Online Auctions
About Us
Contact Us
Auction News
Bidder Sign-In
Search Items
Browse Auctions
Auction Map
Auction Butler
Auctioneers List
Follow Us
Become a Bidder
Become a Seller
Auctioneer Login
Live Auction Software
User Agreement & Privacy Policy
Copyright 1996 - 2019 © iCollector.com Online Auctions


text:
Steven A. Cohen, the founder of the Scottsdale firm Cohen Law, has been accepted into the American Board of Trial Advocates.
ABOTA dedicates itself to the preservation and promotion of the Seventh Amendment, which guarantees the right to civil jury trials, according to a press release.
Members in this group must have tried a minimum of 10 civil jury trials to conclusion. More than 7,600 lawyers and judges are involved in ABOTA chapters in all 50 states.
"ABOTA is one of the nation's most effective organizations in championing the ethics, professionalism and principles of the legal profession, and I'm honored to be part of this select group," Mr. Cohen said.
For more than 40 years, Mr. Cohen has been an advocate for injury victims in Arizona. As a State Bar of Arizona board-certified specialist in injury and wrongful death litigation, he said he is prepared to do whatever it takes to help them with their recovery.
Mr. Cohen is a 1978 graduate of Arizona State University's College of Law. He is a State Bar of Arizona Board-certified specialist in injury and wrongful death litigation and has been a southwest super lawyer for many years.
The Scottsdale Independent publishes a free daily newsletter. A print edition is mailed to 75,000 homes and businesses each month. If you value our journalistic mission, please consider showing us your support.


text:
Now health and safety cut number of holes in chip shop salt shakers
Pot-holed roads, crumbling schools, litter-strewn streets – there's no shortage of problem areas crying out for their attention.
But councils believe they have found a better use for their money: reducing the number of holes in chip shop salt shakers.
Research has suggested that slashing the holes from the traditional 17 to five could cut the amount people sprinkle on their food by more than half.
Trickle or Treat: Chip shop owner Carol Ackerman shows off an old 17-hole shaker and the new model
And so at least six councils have ordered five-hole shakers – at taxpayers' expense – and begun giving them away to chip shops and takeaways in their areas.
Leading the way has been Gateshead Council, which spent 15 days researching the subject of salty takeaways before declaring the new five-hole cellars the solution.
Officers collected information from businesses, obtained samples of fish and chips, measured salt content and 'carried out experiments to determine how the problem of excessive salt being dispensed could be overcome by design'.
They decided that the five-hole pots would reduce the amount of salt being used by more than 60 per cent yet give a 'visually acceptable sprinkling' that would satisfy the customer.
The council commissioned Drywite Ltd – a catering equipment company based in the West Midlands – to make five-hole shakers and bought 1,000 of them at a cost of £2,000, giving them away to fast-food outlets in their areas.
Drywite confirms that it has since received orders for the shakers from at least five other councils, including Rochdale Borough in Greater Manchester.
Another giving the shakers away is Labour-controlled Middlesbrough Council, where the idea has run into fierce criticism.
Cllr Chris Hobson, leader of the Conservatives, said: 'This is just silly, a total waste of money in an area where council tax is very high. I'm all for good health but do they really think they are going to stop people using as much salt simply by putting fewer holes in thecellar? They'll just shake it for longer.'
Beryl Scott, who owns the Chipchase Chippy in Linthorpe in the city, said a council worker had visited the previous week to explain the merits of less salty fish and chips. 'He said he had a salt cellar with five holes to give me free.
I thought it was a joke. It doesn't matter how many holes it has, people are going to put on as much salt as they want.'
Another local chip shop owner, Carol Ackerman, who runs Carol's Plaice in the suburb of Acklam, said: 'People will just put on more salt if they want more.
'In fact, we have had some people unscrewing the lids to do so.'
Gateshead Council defended its decision. A spokesman said: 'Research carried out by us discovered customers were often receiving huge quantities of salt with their fish and chips – up to half their daily allowance. The council was so disturbed it decided to commission a manufacturer to produce a salt shaker with fewer holes, which it distributed free to every fish and chip shop and hot food takeaway in Gateshead.
'We believe the cost to be a small price to pay for potentially saving lives.'
The scheme is being promoted by the Local Authorities Coordinators of Regulatory Services, which is responsible for ensuring councils follow food hygiene rules. A spokesman said: 'Heart disease costs taxpayers £7billion a year so to say that projects such as this are a waste of money is mind-boggling.'
Where do you stand on the salt debate? Tell us your opinion below.
Most watched News videos
- Police shoot and kill man allegedly threatening people with gun
- Mountain biker tries to jump off dry ski slope and lands on his head
- Adorable dog leaps for joy while on walk with owner
- Tiger's ex Elin and NFL beau show off new baby
- Cat gets stuck in pot which gets smashed to set it free
- Turkey's proxy militias film themselves executing Kurdish prisoners
- Sozzled couple holding drinks struggling to walk tumble down stairs
- Two mothers get into fist fight with their babies strapped to them
- Foul-mouthed schoolgirl is exposed in a series of brawl videos
- Dramatic moment teen girls run amok in department store
- Russian woman jailed for 13 years over murder of model sister
- Man escapes from back of NYPD car and is tackled by group of cops


text:
I want my own class with no Miatas allowed.
Crap, just realized that's against the rules.
I want my own class with no Miatas allowed.
Crap, just realized that's against the rules.
Not my circus...not my monkeys...
I'm just glad IT.com just jumped back over eBay in my FireFox feed...
Andy BettencourtNew England Region 188967
SRF and Miatas together at last? Kinda solves my problems (as detailed on the brown board).
Thanks SCCA!
Hero To The Momentum Challenged
Two new classes to consider:
IT-80 - Age of car and driver together must be at least 80 years, No Japanese cars, No FWD. IT prep rules apply. (Reduced entry fee)
IT-30 - Age of car and driver must be less than 30 years, All Japanese cars, No FWD. SM, SSM, or It prep rules apply. (Enhanced entry fee)
Our last Majors at Topeka had SRF and SM together, but there were only 2 SM cars and only one was running competitive lap times. I don't recall if they had a split start or not, but they probably did. http://scca.cdn.racersites.com/prod/...atRace-GP1.pdf
ITB Golf GT
A Majors event with only 2 SM entries? Yeah, that's progress for ya! Keep it up Topeka!!
Houston Region
STU Nissan 240SX
EProd RX7
GA, noting that the STAC is looking for resumes...maybe someone we know would want to get involved in the process(es)...?
Not my circus...not my monkeys...
Greg, I was referring to the HPT race last August with only 14SRF and 2SM.. (See post 392). Both of the strongest spec classes with fewer entries than a December Regional, yet the mothership is touting the Majors program as a resounding success..
With my life and work commitments, I can't volunteer for a committee like that due to the time involved. I had to resign from my local car club stuff that only required 3-4hrs a month because I simply didn't have it to spare. Plus I'm going to Prod. it's cheaper there. :wall:
Houston Region
STU Nissan 240SX
EProd RX7
Sadly, MidDiv is not a useful indicator of the health of Club Racing.
GA, who lived in Texas Region and raced in SWDIV and MidDiv for many years ('90 SSA MidDiv champ, baby!), was in teh very first HPT National, all before deciding to move to New England for better racing pastures...and got stuck here. Oh well, there's always retirement...and I do miss Hallett a lot.
Last edited by Greg Amy; 12-09-2014 at 12:55 PM.
Not my circus...not my monkeys...
There are several tracks in the area, but only a couple that are capable of supporting a Club Race due to lack of facilities/grid/paddock space. We are losing TWS in a few months, but there is talk of another track being built nearby. If it comes through, there will be 9 tracks within a 6 hr tow- 6 or 7 of which could support a club race. Then there's the help of year-round racing if you don't mind racing in a swamp in August! Money is good here with industry and oil, so I'm at a bit of a loss why attendance is lacking here.
Houston Region
STU Nissan 240SX
EProd RX7
Though we're treading off the beaten thread path on this one...here was the state of SWDiv/MWDiv in 1989. This was an email I sent to a guy in regard to the history of my SSA Shelby CSX...
Pulled up the logbook...it was issued 11/11/88, first event
11/12-13/88 at Texas World Speedway. Bill Saunders drove the car in
the SCCA driver's schools the weekends of April 8th 1989 at TWS and
then the following weekend of April 15th at Hallett Motor Speedway,
the car's 4th and 5th race weekends. He apparently passed the second
school, as he finished 1st in the SCCA Regional on Saturday. That
first year the car also raced at Hutchinson Aerodrome in Kansas (an
ex-WW2 Navy airfield), the very first SCCA National at the then-new
Heartland Park in Topeka, another ex-WW2 airfield in Big Spring TX,
the Ponca City (OK) Grand Prix July 4th weekend (probably where that
photo came from), again in Big Spring, won the Solo 1 National
Championship in Showroom Stock A at Hutchinson Aerodrome, finished 23rd
in the 1989 SCCA Runoffs at Road Atlanta, then topped off the year at
the "Bass Ackwards" (reverse direction) annual race at Hallet in
November and the "Early Bird Enduro" at Texas World Speedway in
And it did all this while being driven to each race with tools and
tires in the hatchback...busy year!
Not my circus...not my monkeys...
Hallett gets one SCCA race a year now. TWS gets 1-2, but they're closing forever in May.. Never been to HPT but it's obviously still open. The rest of the tracks and even SOLO sites are long gone.. :\
...Back to your previously scheduled topic...
Houston Region
STU Nissan 240SX
EProd RX7


text:
Live Online Five Animals Qigong Tuition
Instructor Nicola is passionate about sharing qigong with her students. She provides live online private tuition using Skype and/or Facebook video call. Teaching you ancient Taoist movements that will help you towards a regular qigong practice. This type of lessons is personalised for your needs and experience. Beginners are encouraged, this service is open to everyone. Visit the Qigong section in the webstore to book your lesson.
Five Animals Qigong Resources
Sharing with you helpful resources of the Chinese Health Qi Gong Association version of the Five Animal Frolics / Wu Qin Xi Qi Gong set.
Guide to Health Benefits and Traditional Chinese Medicine (TCM)