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Article| November 29 2010
Preferential infection and depletion of Mycobacterium tuberculosis–specific CD4 T cells after HIV-1 infection
Christof Geldmacher,
Christof Geldmacher
Immunology Laboratory
Immunotechnology Section
, and the 3
BSL-3 Core Virology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Department of Infectious Diseases and Tropical Medicine, Klinikum of University of Munich, 80802 Munich, Germany
CORRESPONDENCE Christof Geldmacher: geldmacher@lrz.uni-muenchen.de
Njabulo Ngwenyama,
Njabulo Ngwenyama
Alexandra Schuetz,
Alexandra Schuetz
National Institute for Medical Research-Mbeya Medical Research Programme, Referral Hospital, Mbeya, Tanzania
Constantinos Petrovas,
Constantinos Petrovas
Klaus Reither,
Klaus Reither
Edwin J. Heeregrave,
Edwin J. Heeregrave
Department of Retrovirology, University of Amsterdam, 1105AZ Amsterdam, Netherlands
Joseph P. Casazza,
Joseph P. Casazza
David R. Ambrozak,
David R. Ambrozak
Mark Louder,
Mark Louder
William Ampofo,
William Ampofo
Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
Georgios Pollakis,
Georgios Pollakis
Brenna Hill,
Brenna Hill
Erica Sanga,
Erica Sanga
Elmar Saathoff,
Elmar Saathoff
Leonard Maboko,
Leonard Maboko
Mario Roederer,
Mario Roederer
William A. Paxton,
William A. Paxton
Michael Hoelscher,
Michael Hoelscher
Richard A. Koup
Christof Geldmacher and Njabulo Ngwenyama contributed equally to this paper.
C. Geldmacher's present address is Institute for Tropical Medicine and Infectious Diseases, Ludwig-Maximilians University of Munich, 80802 Muenchen, Germany.
A. Schuetz's present address is Henry M. Jackson Foundation for the Advancement of Military Medicine, Walter Reed Program, US Army Medical Component of the Armed Forces Research Institute of the Medical Sciences, Dept. of Retrovirology, Bangkok 10400, Thailand.
AZT
azidothymidine
Brefeldin A
CFP10
culture filtrate protein 10
complete media
ESAT6
early secretory antigenic target 6
phytohemagglutinin
purified protein derivative
region of difference 1
staphylococcal enterotoxin B
simian immunodeficiency virus
Received: January 12 2010
J Exp Med (2010) 207 (13): 2869–2881.
Christof Geldmacher, Njabulo Ngwenyama, Alexandra Schuetz, Constantinos Petrovas, Klaus Reither, Edwin J. Heeregrave, Joseph P. Casazza, David R. Ambrozak, Mark Louder, William Ampofo, Georgios Pollakis, Brenna Hill, Erica Sanga, Elmar Saathoff, Leonard Maboko, Mario Roederer, William A. Paxton, Michael Hoelscher, Richard A. Koup; Preferential infection and depletion of Mycobacterium tuberculosis–specific CD4 T cells after HIV-1 infection. J Exp Med 20 December 2010; 207 (13): 2869–2881. doi: https://doi.org/10.1084/jem.20100090
HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common opportunistic pathogens, cytomegalovirus (CMV) and Mycobacterium tuberculosis (MTB). CMV-specific CD4 T cells persisted after HIV infection, whereas MTB-specific CD4 T cells were depleted rapidly. CMV-specific CD4 T cells expressed a mature phenotype and produced very little IL-2, but large amounts of MIP-1β. In contrast, MTB-specific CD4 T cells were less mature, and most produced IL-2 but not MIP-1β. Staphylococcal enterotoxin B–stimulated IL-2–producing cells were more susceptible to HIV infection in vitro than MIP-1β–producing cells. Moreover, IL-2 production was associated with expression of CD25, and neutralization of IL-2 completely abrogated productive HIV infection in vitro. HIV DNA was found to be most abundant in IL-2–producing cells, and least abundant in MIP-1β–producing MTB-specific CD4 T cells from HIV-infected subjects with active tuberculosis. These data support the hypothesis that differences in function affect the susceptibility of pathogen-specific CD4 T cells to HIV infection and depletion in vivo, providing a potential mechanism to explain the rapid loss of MTB-specific CD4 T cells after HIV infection.
Microbial Pathogenesis & Host Defense
HIV infection is characterized by the progressive depletion of CD4 T helper cells, eventually leading to AIDS and the onset of various opportunistic diseases. Highly active antiretroviral therapy interrupts HIV replication and leads to increases in CD4 T cell numbers and function, with associated clearance of many opportunistic infections. Data indicate that ongoing viral replication within CD4 T cells is among the most important parameters driving the massive, systemic depletion of memory CD4 T cells during acute immunodeficiency virus infection (Li et al., 2005; Mattapallil et al., 2005) and the subsequent slow depletion of CD4 T cells during chronic disease. Although CD4 T cell–mediated delayed type hypersensitivity reactions are lost during disease progression (Blatt et al., 1993; Raszka et al., 1996), it is largely unclear whether depletion of particular pathogen-specific CD4 T cells equates with susceptibility to given opportunistic infectious diseases. Some data suggest that this might be the case; e.g., cytomegalovirus (CMV) end organ disease is associated with and preceded by the loss of CMV-specific CD4 T cell responses (Komanduri et al., 1998; Bronke et al., 2005). Whether different pathogen-specific CD4 T cell populations are being differentially affected by HIV and what pathogenic mechanisms contribute to the depletion of individual pathogen-specific CD4 T cells is unknown.
Pathogen-specific α/β (αβ) CD4 T cells play a central role in the orchestration of adaptive immune responses and are important for protective immunity to many microbial pathogens, including Mycobacterium tuberculosis (MTB; Caruso et al., 1999; Gallegos et al., 2008). CD4 T cells are a crucial component of the response to acute MTB infection in the mouse model (Mogues et al., 2001). Upon antigen recognition, MTB-specific CD4 T cells secrete IFN-γ and TNF, which activate infected macrophages and contribute to the containment of intraendosomal bacilli (Cooper et al., 1993; Flynn et al., 1995; Kaufmann and McMichael, 2005) and to the formation of bactericidal granulomas (Ray et al., 2009). Hence, MTB-specific CD4 T cells are thought to be of central importance in the efficient control of MTB infection and prevention of further dissemination to extrapulmonary sites.
Active pulmonary and extrapulmonary tuberculosis (TB) are among the most commonly observed opportunistic infectious diseases in HIV-infected subjects within MTB endemic areas, and pulmonary TB is frequently the first manifestation of AIDS in such regions (http://www.who.int/tb/challenges/hiv/faq/en/, accessed on Dec third 2009). Within areas of high TB incidence, the risk of developing active TB is significantly increased, even during the first year after HIV infection when total CD4 T cell counts are still quite high (Kaufmann and McMichael, 2005; Sonnenberg et al., 2005). TB was shown to be the cause of death in almost 50% of HIV seropositive South African gold miners (Murray et al., 2007). Furthermore, evidence of active and disseminated TB was found in almost 50% of post-mortem autopsies conducted on HIV-infected Kenyan subjects (Rana et al., 2000). In the absence of HIV infection or other immunodeficiencies, MTB is reasonably well controlled, and only ∼10% of MTB-exposed individuals develop active TB disease. Thus, the dramatic increase in active TB associated with HIV infection suggests that MTB-specific immunity might be particularly vulnerable to HIV-associated immune damage.
In stark contrast to pulmonary TB, which frequently occurs in HIV-infected subjects with relatively high CD4 T cell counts, CMV-associated end organ disease typically affects AIDS patients only after CD4 counts have fallen to very low levels. Although these pathogens differ substantially, MTB and CMV share a range of similarities. Both cause persistent or latent infections that are tightly controlled by the adaptive immune system in healthy individuals, but cause life threatening disease in immunocompromised states. Importantly, pathogen-specific CD4 T cells play important roles in the control of both infections (Komanduri et al., 1998, 2001; Caruso et al., 1999; Gamadia et al., 2003; Bronke et al., 2005; Gallegos et al., 2008).
MTB-specific CD4 T cell responses are depleted in peripheral blood early after HIV infection, whereas they do not change appreciably over a 6–12-mo time interval in HIV-negative subjects with latent MTB infection (Geldmacher et al., 2008). During chronic HIV infection, MTB-specific CD4 T cell responses are significantly decreased in the periphery and bronchoalveolar lavage (BAL; Geldmacher et al., 2008; Kalsdorf et al., 2009). In contrast, CMV-specific CD4 T cells are often detectable even late into chronic HIV infection (Waldrop et al., 1997). This difference provides an opportunity to study cellular parameters associated with the rapid depletion or persistence of pathogen-specific CD4 T cells after HIV infection and to further elucidate the mechanisms underlying the differential rates of depletion of various pathogen-specific CD4 T cells. A better understanding of such mechanisms might help to elucidate general mechanisms of HIV pathology and to define parameters that can protect vaccine-induced HIV-specific CD4 T cells from preferential infection and depletion upon exposure to HIV.
MTB-specific and CMV-specific CD4 T cell responses after HIV infection
To determine the effect of HIV on different pathogen-specific CD4 T cells in the periphery, we first compared the frequencies of MTB- and CMV-specific CD4 T cell responses shortly before, early after, and during chronic HIV infection in subjects without evidence of active TB or CMV disease. In 5 Tanzanian subjects who acquired HIV infection during the study, MTB- and CMV-specific responses were detectable at 3 mo before the first HIV seropositive study visit. During this baseline visit, the median frequencies of MTB- and CMV-specific memory CD4 T cells were 0.18% (range, 0.1–0.8) and 0.51% (range, 0.36–2.6), respectively. The median frequency of MTB-specific CD4 T cells decreased by 89.4% (Fig. 1) within 6–12 mo after HIV seroconversion. MTB-specific CD4 T cells completely disappeared in four of five subjects, and decreased by more than fivefold in the fifth subject. We had previously demonstrated that MTB-specific CD4 T cells do not change appreciably over a 6–12-mo time interval in HIV− subjects with latent MTB infection (Geldmacher et al., 2008). In contrast, CMV-specific CD4 T cells were still detectable after 6–12 mo in all subjects, with a median decrease of only 40.6%, despite recent HIV infection. In four subjects, the frequencies of CMV-specific CD4 T cells were also determined during the first HIV seropositive visit and/or at 3 or 6 mo thereafter, but were always detectable (unpublished data). The absolute decline in MTB- and CMV-specific CD4 T cells after HIV infection was also quantified in subjects with available CD4 T cell counts before and early after HIV infection. MTB-specific CD4 T cells declined from a median of 1,092 cells/ml (n = 4; range, 572–2,525) to a median of 29 cells/ml (range, 0–427), and CMV-specific CD4 T cells declined from a median of 3,298 cells/ml (n = 5; range, 2,316–6,300) to a median of 1,184 cells/ml (range, 1,061–5,457). Collectively, these data demonstrate that the rate of in vivo CD4 T cell depletion early after HIV infection can differ depending on the antigen specificity of the response.
The effect of HIV infection on the frequencies of MTB- and CMV-specific CD4 T cells was also determined in a cross-sectional analysis of HIV-infected and uninfected Tanzanian subjects (n = 17 HIV+, 17 HIV−; Fig. 1, B and C). All subjects were clinically asymptomatic for MTB or CMV infection. HIV-infected subjects had a median CD4 T cell count of 640 cells/µl (range: 109–758). In line with previous studies (Waldrop et al., 1997), CMV-specific CD4 T cells were detectable in most samples, and the median magnitude of the response was actually higher in HIV+ than HIV− subjects (3.32 versus 1.72%, respectively). In contrast, HIV infection was associated with significantly reduced magnitudes (P = 0.009) and reduced prevalence of detectable MTB-specific responses. 6 of 17 HIV+ subjects had detectable MTB-specific CD4 T cell responses compared with 14 of 17 in HIV− subjects. Collectively, these data demonstrate that early and chronic untreated HIV infection is associated with severe depletion of MTB-specific CD4 T cell responses, whereas CMV-specific CD4 T cell responses tend to persist. These data suggest that MTB-specific CD4 T cells are more susceptible than CMV-specific CD4 T cells to in vivo HIV infection and deletion.
Phenotype and function of MTB- and CMV-specific CD4 T cells
To investigate factors that might be associated with the different rates of CD4 T cell decline, we first assessed expression of the HIV coreceptor, CCR5, on MTB- and CMV-specific CD4 T cells. Although CCR5 was present on the CD4 T cells of both specificities, there was no difference in the expression of CCR5 (percentage or mean fluorescent intensity) between CD4 T cells of the two specificities (unpublished data).
We next examined multiple other surface maturation markers and several functional characteristics within the following groups of pathogen-specific CD4 T cells: (a) MTB- and CMV-specific CD4 T cells in HIV− subjects in the absence of active TB disease and (b) MTB-specific CD4 T cells in association with active TB disease in HIV+ subjects (Fig. 2). Cellular maturation was studied using the differentiation markers CD27, CD45RO, and CD57, which discriminate between naive (CD27+CD45RO−), central memory-like (CD27+CD45RO+), effector memory-like (CD27−CD45RO+), and terminally differentiated (CD57+) CD4 T cells (Brenchley et al., 2003).
In HIV− subjects with latent TB, the majority of MTB-specific CD4 T cells had a CD27+CD45RO+ phenotype (median 64%; Fig. 2 A). A minority had a CD27−CD45RO+ phenotype (median 36%). CD57 expression was not detectable or insignificant. In contrast, MTB-specific CD4 T cells in HIV+ subjects with active TB were phenotypically distinct and were characterized by down-regulation of CD27 (median 83% were CD45RO+CD27−; P < 0.001) and increased expression of CD57 (median 9.5%; P < 0.05). CMV-specific CD4 T cells also lacked CD27 expression (median 85.6% were CD45RO+CD27−) and showed even more frequent expression of CD57 (median 48.4%).
To determine whether cell maturation was associated with functional changes within pathogen-specific CD4 T cells, we analyzed expression of IFN-γ, IL-2, TNF, and MIP-1β within the three different groups of CD4 T cells and delineated the functions according to their maturational phenotype. As shown in Fig. 2 B, diametrically opposed patterns of IL-2 and MIP-1β production were detected in MTB-specific CD4 T cells among HIV− TB asymptomatic subjects and CMV-specific CD4 T cells (right). Large fractions of CMV-specific CD4 T cells produced MIP-1β (median, 62%), but few produced IL-2 (median, 20%). In contrast, most MTB-specific CD4 T cells in asymptomatic infection produced IL-2 (P < 0.001; median, 54%), but not MIP-1β (P < 0.001; median, 3%). Differences in MIP-1β and IL-2 production occurred throughout the 16 possible cytokine combinations (bottom). No such differences were detected for TNF or IFN-γ production (unpublished data). MTB-specific CD4 T cells in HIV+ subjects with active TB were functionally distinct from those in HIV− subjects during asymptomatic TB infection. The median fraction of MIP-1β+ cells was higher (29%; P < 0.05), whereas the median fraction of IL-2+ cells was lower (32%; P < 0.05) in HIV+ subjects with active TB as compared with the response in HIV− subjects with latent TB. Most notably, the subset of three functional cells that produced MIP-1β, IFN-γ, and TNF, but no IL-2, which is characteristic of the CMV-specific CD4 T cell response, was virtually absent from MTB-specific CD4 T cells during latent TB, but present during active TB.
Whether these distinct functional properties were associated with different cellular maturation was then analyzed by comparing pathogen-specific IL-2 and MIP-1β production by CD27+CD45RO+, CD27−CD45RO+CD57−, and CD27−CD45RO+CD57+ CD4 T cells. A representative dot plot (left) for IL-2 and MIP-1β production for each group and for all studied responses (right) is shown in Fig. 2 C. Independent of the pathogen specificity, a CD45RO+CD27− phenotype was associated with more MIP-1β+ and fewer IL-2+, cells. Large fractions of CD27−CD45RO+CD57+ CD4 T cells were only present within the CMV-specific response, and almost all expressed MIP-1β exclusively. Virtually none of the CD27−CD45RO+CD57+ cells produced IL-2, irrespective of specificity. Together, these data suggest that the capacity to produce IL-2 is reduced, whereas the capacity to produce MIP-1β is increased, with maturation of pathogen-specific CD4 T cells.
Effect of HIV infection on CD4 T cell function and phenotypes in vivo
Less IL-2 production and greater MIP-1β production among MTB-specific CD4 T cells in HIV+ versus HIV− subjects, and between MTB and CMV-specific CD4 T cells (Fig. 2), is consistent with the hypothesis that HIV preferentially infects and depletes IL-2–producing CD4 T cells and is partially inhibited from infecting and depleting MIP-1β–producing CD4 T cells in vivo. We further addressed this possibility by comparing the proportion of CMV-specific, cytokine-producing cells among HIV+ and HIV− subjects (Fig. 3). There was no difference in the proportion of IFN-γ– or TNF-producing CMV-specific CD4 T cells in HIV+ and HIV− subjects. However, there was a significantly smaller proportion of IL-2–producing cells (P < 0.05; Fig. 3) and a significantly greater proportion of MIP-1β–producing cells (P < 0.05, Fig. 3) in HIV+ versus HIV− subjects. This difference was reflected in cells that produced MIP-1β but not IL-2 and produced IL-2 but not MIP-1β, but not in the cells that produced both effector molecules simultaneously (Fig. 3, right). Hypothetically, the decline in MTB-specific CD4 T cells could also be explained by selective anergy within the CD27+ memory CD4 compartment or selective depletion of this compartment after HIV infection. However, the functional capacity of the CD27+ memory CD4 compartment upon staphylococcal enterotoxin B (SEB) stimulation was not compromised after HIV infection and the proportion of CD27+ memory CD4 compartment remained constant after HIV infection in six HIV seroconverters (unpublished data). These data are therefore consistent with the hypothesis that with HIV infection in vivo, IL-2–producing cells are more likely to get infected and depleted than MIP-1β–producing cells.
IL-2 dependence of HIV infection in vitro
The in vitro inhibitory effects of CCR5 ligands on HIV infection have been previously described (Cocchi et al., 1995; Kinter et al., 1996; Paxton et al., 1996). To explore whether endogenous IL-2 production augments in vitro HIV infection, we performed HIV infection experiments in the presence or absence of a neutralizing anti–IL-2 antibody. SEB-stimulated PBMCs were infected with the CCR5-tropic HIV strain BAL, and CD4 T cells were analyzed for HIV p24 content in the context of cell division and CD25 and CCR5 surface expression (in the absence of exogenously added IL-2). We also attempted stimulation with purified protein derivative (PPD) in this experimental set up. However, the frequency of responding cells was so low that there were too few events in which to accurately measure HIV infection. The p24 staining in this system was the result of active viral replication, and not retained p24 from the virus inoculum, as it was inhibited by the addition of the reverse transcription inhibitor azidothymidine (AZT; Fig. 4 A). In the absence of the IL-2 neutralizing antibody, 48% of dividing cells (CFSElow), but only 2.2% of the cells that had not undergone cell division were p24+ (Fig. 4 B, top). Virtually all of these p24+ cells expressed CD25, demonstrating that productive HIV infection occurred within cells that could respond to IL-2 signaling. Neutralization of IL-2 resulted in a 12.5-fold reduction of p24+ cells and completely abrogated cell division (Fig. 4 B, bottom). The fraction of CD25+ cells was reduced by 60% (Fig. 4 B, bottom), whereas the fraction of CCR5+ cells was unaffected by the anti–IL-2 treatment, indicating that neutralization of IL-2 did not affect HIV replication through modulation of the coreceptor, but by affecting post-entry events associated with cell division. These data demonstrate that productive HIV-infection of SEB-stimulated CD4 T cells is associated with CD25 expression, cell cycle induction, and the production of IL-2 upon TCR engagement.
We next analyzed the expression of CD25 on IL-2+MIP-1β− and IL-2−MIP-1β+ CD4 T cells after 6 h SEB stimulation. Fig. 5 A shows a single experiment, and Fig. 5 B shows the composite results from seven experiments. These data demonstrate that a median of 39.5% of IL-2+MIP-1β−, but only 6.6% of MIP-1β+IL-2− CD4 T cells express CD25 after stimulation. The expression of CD25 on the MIP-1β+IL-2− CD4 T cells is the same as on all other nonresponding memory CD4 T cells, suggesting that IL-2 is specifically and intimately linked to expression of its high-affinity receptor, CD25.
We next analyzed in vitro HIV infection of SEB-stimulated CD4 T cells according to their expression pattern of MIP-1β, IFN-γ, and IL-2 (Fig. 6). Within the responding CD4 T cell populations, we were able to gate upon cells with cytokine profiles that match either CMV- (MIP-1β+IL-2−) or MTB-specific (MIP-1β−IL-2+) CD4 T cells. CD4 T cells with a cytokine profile matching that of MTB-specific T cells had higher HIV infection rates as compared with those matching CMV-specific T cells (P = 0.03).
Cytokine expression delineates in vivo infection history of MTB-specific T cells
To determine in vivo cellular infection rates within MTB-specific and nonspecific memory CD4 T cells, we stimulated PBMCs from HIV/MTB coinfected subjects with a mix of PPD, early secretory antigenic target 6 (ESAT6), and culture filtrate protein 10 (CFP10), sorted the CD4 T cells according to cytokine and chemokine expression (Fig. 7 A), and quantified the HIV gag DNA within the different subsets. Despite relatively low numbers of sorted cytokine/chemokine+ cells, we were able to quantify proviral DNA in 16 responding MTB-specific CD4 T cell populations from 10 HIV-infected subjects with active TB. We first compared the number of HIV gag DNA copies within MTB-specific, cytokine/chemokine+ CD4 T cell samples with the nonresponding CD4 memory T cell compartment (CD27+CD45RO+, CD27−CD45RO+, and CD27−CD45RO− subsets). In 9 of 10 subjects, the number of HIV gag DNA copies detected in MTB-specific CD4 T cells was higher compared with total memory CD4 T cells (median 2.5-fold increase; P < 0.01; Fig. 7 C). Next, we determined whether MTB-specific CD4 T cells expressing different patterns of IL-2 and MIP-1β contained different amounts of HIV gag DNA. MTB-specific CD4 T cells that exclusively expressed either MIP-1β or IL-2 were virtually nonexistent. The median number of HIV gag DNA copies per 10,000 cells was 1,426.5 for IFNγ+IL-2+MIP-1β−TNF+ (range 109–127,500 mean), 538 for IFNγ+IL-2−MIP-1β−TNF+ (range, 29–34,167), 315 for MIP-1β+TNF+ (range, 278–6,136), and 268 for cytokine negative memory CD4 T cells (range, 8–19,370). To reduce the inherent patient-to-patient variability in the HIV gag DNA copy numbers, and overcome the fact that many of the sorted samples contained too few cells to register a signal in the PCR assay, the results were expressed as a ratio of HIV gag DNA copies in cytokine+/cytokine− cells (Fig. 7 B). Consistent with the aforementioned raw data calculations, the IL-2–producing MTB-specific CD4 T cells tended to harbor greater numbers of HIV gag copies per cell as compared with the IL-2− and MIP-1β+ cells, although this trend was not statistically significant within this small number of subjects. Together, these data support the hypothesis that the functional characteristics, and in particular the capacity to secrete IL-2 but not MIP-1β upon MTB-specific stimulation, are associated with increased susceptibility of MTB-specific CD4 T cells to HIV infection.
We have previously demonstrated that MTB-specific CD4 T cells are depleted from the periphery early after HIV infection, whereas in HIV− subjects the frequencies of MTB-specific CD4 T cells do not change appreciably over time (Geldmacher et al., 2008). In contrast, CMV-specific CD4 T cells are frequently detectable in the periphery during chronic HIV infection (Waldrop et al., 1997) and contain comparatively little proviral DNA (Douek et al., 2002). In this study, we detected significantly higher levels of HIV gag DNA in MTB-specific CD4 T cells than in total memory CD4 cells in the periphery. The higher levels of HIV gag DNA within MTB-specific CD4 T cells (ratio of 2.5 to total memory CD4 T cells) are comparable to those observed in HIV-specific CD4 T cells (ratio 3.4; Douek et al., 2002). Brenchley et al. (2006) have previously shown that HIV-specific CD4 T cells are present at very low frequencies in most HIV+ individuals and that direct HIV infection of virus-specific CD4 T cells reduces their life span in vivo. In this context, these data support the hypothesis that direct HIV infection of MTB-specific CD4 T cells contributes to their depletion, possibly compromising the generation of effective immune responses to MTB in HIV-infected subjects. We thus explored which functional and phenotypic characteristics might account for the higher infection rates and more rapid depletion of peripheral MTB-specific CD4 T cells after HIV infection and tested the hypothesis that differences in specific functions might contribute to differential HIV infection of antigen-specific CD4 T cells in vivo.
We have previously shown that MTB-specific CD4 T cells express comparatively high levels of the HIV-coreceptor CCR5. However, CCR5 expression was similar on MTB- and CMV-specific CD4 T cells (unpublished data) indicating that coreceptor expression alone could not account for any difference in rates of depletion after HIV infection between MTB- and CMV-specific CD4 T cells. Instead, we found significant differences in the capacity of MTB- and CMV-specific CD4 T cells to produce IL-2 and the CCR5 ligand MIP-1β. CMV-specific CD4 T cells tended to produce MIP-1β but not IL-2, and displayed an effector memory phenotype and this functional profile that was even more pronounced during chronic HIV infection, whereas MTB-specific CD4 T cells produced IL-2 but not MIP-1β, and were less mature. Cells producing IL-2 in the absence of MIP-1β were more susceptible to productive HIV infection within SEB-activated CD4 T cells in vitro (Fig. 6) and this functional profile within MTB-specific CD4 T cells was associated with a greater frequency of HIV gag DNA in vivo (Fig. 7). These observations together with the striking differences in the depletion dynamics between MTB- and CMV-specific CD4 T cells early after HIV infection suggest that these differences in function contribute to differential cellular susceptibility to HIV infection and depletion of these two cell populations, and the associated differences in disease onset caused by these opportunistic pathogens.
The capacity of CCR5 ligands to block HIV replication in vitro is well established (Cocchi et al., 1995, Kinter et al., 1996). Indeed, CD4 T cells with a relative resistance to CCR5-tropic HIV strains are capable of producing comparatively high amounts of such CCR5 ligands, including MIP-1β (Paxton et al., 1996). We have recently shown that CMV-specific CD4 T cells that produce MIP-1β and MIP-1α contain ∼10-fold less HIV gag DNA than CMV-specific CD4 T cells that do not produce these chemokines (Casazza et al., 2009). These data indicate that autocrine production of MIP-1β protects CD4 T cells from HIV infection in vivo. Thus, the lack of MIP-1β production by the less mature MTB-specific CD4 T cells might contribute to their increased susceptibility to HIV infection and subsequent depletion. In contrast, the production of MIP-1β by the more mature CMV-specific CD4 T cells is likely to contribute to their relative resistance to HIV infection (Douek et al., 2002; Casazza et al., 2009), and thus to their persistence after HIV infection. Our observation that HIV infection is associated with further increased proportions of MIP-1β+ but decreased proportions of IL-2+ CMV-specific CD4 T cells is in line with these findings and with our hypothesis that the functional profile of pathogen-specific CD4 T cells might influence their susceptibility to HIV. Indeed, as a common theme, the tendency of more differentiated CD4 T cells to produce MIP-1β could explain the previously described low HIV infection rates within CD57+ CD4 T cells in vivo (Brenchley et al., 2003).
IL-2 signaling pathway and HIV infection
Whereas MTB-specific CD4 T cells lacked the capacity to produce MIP-1β in individuals with latent MTB infection, they had a remarkable capacity to produce IL-2. IL-2 and its high-affinity IL-2 receptor α-chain CD25, whose expression rapidly increases upon T cell receptor engagement, are important in driving T cells into the cell cycle. In addition to being critical for T cell proliferation, IL-2 may also positively impact upon HIV reverse transcription (Zack et al., 1990) by increasing the concentration of dNTPs in CD25+ CD4 T cells. Indeed, CD25 expression and productive HIV infection appear to be linked; in vitro depletion of CD25+ CD4 T cells efficiently blocks viral replication (Finberg et al., 1991;, Ramilo et al., 1993; Chou et al., 1997). CD25 expression on CD4 T cells correlates with PPD-induced HIV replication (Goletti et al., 1996), and it was recently demonstrated that productive HIV-1 infection within lymphoid tissue explants occurred predominantly in CD4 T cells expressing CD25 (Biancotto et al., 2008). In vivo, CD25+ CD4 T cells contain up to sevenfold more immunodeficiency virus during acute infection, a phenomenon that was particularly apparent within lymph nodes (Mexas et al., 2008). Our observations that in vitro neutralization of IL-2 reduced CD25 up-regulation, abrogated antigen-specific CD4 T cell proliferation, and almost completely abrogated productive HIV infection of CD4 T cells supports a central role for the IL-2–CD25 signaling pathway in the productive infection of antigen-specific CD4 T cells. Our finding that peripheral MTB-specific CD4 T cells with the capacity to produce IL-2, but not MIP-1β, contained the highest amount of HIV gag DNA in vivo supports the conclusion that IL-2 expression by MTB-specific CD4 T cells leads to their increased susceptibility to HIV infection and depletion. However, despite the large numbers of PBMCs used for a single experiment (3 × 107), the number of sorted MTB-specific CD4 T cells with different functional profiles was often too low to detect HIV DNA, and larger studies will be needed to expand upon this finding. The capacity to produce IL-2 in the absence of MIP-1β might create a microenvironment that very efficiently drives the replication of HIV within MTB-specific CD4 T cells. Indeed, the enormous potential of MTB-specific immune responses to contribute to an explosive replication of a single virus quasi species in vivo has been demonstrated within simian immunodeficiency virus (SIV)–infected Rhesus macaques; up to 35% of viral quasi species in PBMCs were closely related to the single major species that was isolated at the site of the tuberculin skin test (Cheynier et al., 1998).
Impact of function and phenotype on HIV infection
Activated, CD25-expressing CD4 T cells that are HIV infected have been shown to preferentially undergo apoptosis in lymphoid tissue explants (Biancotto et al., 2008), and SIV infection of CD4 T cells at mucosal sites can reach extremely high frequencies during acute infection (Mattapallil et al., 2005). Our observations support the hypothesis that MTB-specific IL-2+MIP-1β− CD4 T cells might represent a pool of CD4 T cells that are highly susceptible to HIV infection and may become rapidly depleted upon HIV infection before they can further differentiate into effector cells, a general mechanism that has been proposed previously for the depletion of memory CD4 T cells (Grossman et al., 2006; Okoye et al., 2007). In this scenario, the few MTB-specific CD4 T cells that avoid HIV infection could go on to mature further, and in the process gain expression of MIP-1β and lose IL-2 expression, simultaneously making them less susceptible to HIV infection. The resulting shift from IL-2 expression to MIP-1β expression concomitant with loss of CD27 and gain of CD57 expression that is predicted by this model is exactly what we observed in MTB- and CMV-specific CD4 T cells in individuals who were infected with HIV (Fig. 2 C). Collectively, our observations support the hypothesis that antigen-specific IL-2 production, particularly in the absence of CCR5 ligand MIP-1β, contributes to the productive HIV infection and subsequent depletion of MTB-specific CD4 T cells early after HIV infection.
During clinically latent TB, the majority of MTB-specific CD4 T cells expressed CD27 and can be considered as having a central memory-like phenotype (CCR7 was not measured); however, they also expressed higher levels of CCR5 compared with the total memory compartment (Geldmacher et al., 2008). In contrast, CMV-specific CD4 T cells had a highly differentiated effector memory-like phenotype. HIV infection was associated with a significantly increased fraction of senescent, CD57+ cells (Brenchley et al., 2004) and MIP-1β+ cells, and a reduced fraction of cells with the capacity to produce IL-2. Similarly, in HIV patients with active TB, MTB-specific CD4 T cells were characterized by a more mature CD27low phenotype, a detectable fraction of CD57+ and MIP-1β+ CD4 T cells, and a reduced capacity to produce IL-2. These differences might also be driven by the strength and persistence of T cell receptor engagement and thus different patterns of pathogen reactivation might contribute to the phenotype and function of pathogen-specific CD4 T cell responses and also to their persistence or reappearance after HIV infection; indeed CMV viral replication can be detected in almost ∼20% of HIV infected subjects (Griffin et al., 2008, Slyker et al., 2009) and MTB reactivation might account for the change in phenotype and function in subjects with active as compared with latent TB (Streitz et al., 2007). However, CD4 T cells targeting cleared antigens, such as the flu or the tetanus toxin, and with the capacity to produce IL-2 are also disproportionally depleted (Tilton et al., 2007) upon antiretroviral treatment interruption, suggesting that this phenomenon cannot be solely explained by enhanced stimulation, TCR driven maturation and associated changes in functionality. Thus, increased HIV infection among those cells still capable of producing IL-2 might preferentially deplete these less mature, central memory-like CD4 T cells.
Because our study focuses on the phenotype and function of antigen-specific cells within the periphery, we are unable to address the impact of antigen-specific T cell functional changes, as they occur after antigen exposure in other tissues, nor can we rule out specific migration of cells with certain phenotypes or functions as an explanation for the changes we observe. However, other published data suggest that MTB-specific CD4 T cells are depleted also from the BAL of HIV infected, TB asymptomatic subjects from a TB endemic area (Kalsdorf et al., 2009).
Implications for vaccines
Preferential infection of vaccine-induced, HIV-specific CD4 T cells might reduce efficiency of vaccine-induced immunity. Our results help to define cellular parameters associated with HIV infection and either depletion or persistence of antigen-specific CD4 T cells. These findings could help define the type of CD4 response one would hope to induce with a HIV vaccine. Indeed, just recently it has been shown that vaccination with replication competent, recombinant CMV was able to induce differentiated, MIP-1β–producing HIV-specific T cells that were associated with protection against SIV challenge (Hansen et al., 2009).
In conclusion, our data suggest that specific functional and maturational characteristics of MTB-specific CD4 T cells might contribute to increased HIV susceptibility and their subsequent depletion after HIV infection. Strong antigenic stimulation during active TB might obscure this depleting effect, despite high infection (and turnover) rates of MTB-specific memory CD4 T cells, and at the same time might drive phenotypic and functional changes that confer partial cellular resistance to HIV infection. These results suggest that MTB-specific adaptive immunity is particularly vulnerable to HIV-associated immune damage.
Study subjects.
Subjects from three cohorts were included in this study. Most were part of a large, well-characterized, high-risk cohort of female bar workers (HISIS) enrolled in a prospective study of HIV-1 infection in the Mbeya region of Southwest Tanzania, which is described in detail elsewhere (Riedner et al., 2003; Herbinger et al., 2006). Subjects with evidence of active TB were enrolled at the NIMR-Mbeya Medical Research Program (NIMR-MMRP) Tuberculosis Clinic in collaboration with the National Tuberculosis and Leprosy Program of Tanzania (NTLP). The purpose and the procedures of the study were explained thoroughly to potential participants. Only persons who gave voluntarily written informed consent in the presence of a witness were enrolled. Acid Fast Bacilli smear positive TB patients were referred to the MMRP Tuberculosis clinic. Active TB was diagnosed by examination and culture (BACTEC MGIT; BD) of three separate sputum samples per patient. HIV status was determined using HIV 1/2 STAT-PAK (Chembio Diagnostic Systems) and positive results were confirmed using Enzygnost Anti-HIV 1/2 Plus ELISA (Siemens). Patients eligible for TB treatment were referred to the NTLP for treatment according to national guidelines. Patients diagnosed with HIV infection were referred to the Southern Highland Care and Treatment Program. None of the subjects evaluated in the current study were on active antiretroviral therapy.
In addition, blood was obtained from HIV-positive antiretroviral therapy naive Ghanaian patients with suspected TB attending the HIV clinic of Koforidua Hospital, Ghana, after informed consent. Ethical clearance was granted by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, NMIMR-IRB CPN 039/06-07. HIV infection was confirmed using the Determine HIV-1/2 test (Abbott) and the First Response HIV 1–2.0 test (Premier Medical Corporation Ltd).
PBMCs from these patient cohorts were isolated, cryopreserved, stored in liquid nitrogen, and used in subsequent immunological assays. Use of these patient materials was reviewed and approved by the ethics committees of all partners in compliance with national guidelines and institutional policies.
Conjugated antibodies.
The following antibodies were used: IFN-γ–FITC, IL-2-allophycocyanin (APC), MIP-1β-PE, CD3-Cy7APC, CCR5-Cy7PE and CD25-Cy5PE (BD), CD27-Cy5PE and CD45RO-Texas red-PE (Beckman Coulter), and CD4-PECy5.5 (Invitrogen) and anti-HIV p24/p55 antibody (PE or FITC; clone KC57; Beckman Coulter). CD8-quantum dot (QD) 655, CD57-QD565, and TNF-Alexa Fluor 680 were conjugated in accordance with standard protocols (http://drmr.com/abcon/index.html).
Antigens.
For cell stimulations, PPD (Statens Serum Institut) was used at a final concentration of 10 µg/ml. 2 µl of undiluted CMV grade 2 antigen (Microbix) was used to stimulate PBMC. Peptides 15 aa in length and overlapping by 11 aa were designed for ESAT6 (AF420491.1) and CFP10 (AAC83445) by using the PeptGen peptide generator from the HIV Molecular Immunology Database (http://www.hiv.lanl.gov/content/immunology/) and were used at a final concentration of 2 µg/ml/peptide. For sorting experiments, MTB-specific CD4 T cells were stimulated overnight with a mix of PPD, ESAT6, and CFP10 peptides to maximize detection of the MTB-specific T cell population.
Stimulation and flow cytometric analysis.
Previously cryopreserved PBMCs were recovered by thawing and washing twice in Benzonase (4 µl of 25 kU Benzonase in 20 ml; Novagen) containing 37°C complete media (CM; 10% heat inactivated fetal calf serum, 100 U/ml penicillin G, 100U/ml streptomycin sulfate, and 1.7 mm sodium glutamine). Cell stimulation was performed in CM in the presence of 1 µg/ml each of anti-CD28 and anti-CD49d (BD). Duration of stimulation varied in different assays. For the detection of MTB- and CMV-specific CD4 T cell responses and analysis of their functionality and cell surface phenotype, PBMCs were stimulated with specific antigens for 2 h before adding Brefeldin A (BFA; Sigma-Aldrich), and then further incubated overnight (14–18 h; 6 h for analysis of CD25 expression) at 37°C. For in vitro HIV infection experiments, PBMCs were stimulated for 48 h and BFA was added 4 h before flow cytometric analysis. Staining was performed using a modified version of a previously described method (Betts et al., 2006). After stimulation, PBMCs were washed once with PBS and stained with Vivid/Aqua (Invitrogen; Perfetto et al., 2006). Anti-CCR5 antibody was then added for 10 min at room temperature in the dark. Subsequently, surface proteins were stained for 20 min. The cells were permeabilized using the Cytofix/Cytoperm kit (BD), after which they were stained for CD3, IFN-γ, MIP-1β, IL-2, and TNF. Cells were then washed and fixed with 1% paraformaldehyde. Cells were analyzed with a modified flow cytometer (LSR II; BD). Between 300,000 and 1,000,000 total events were collected from each sample. Electronic compensation was conducted with antibody capture beads (BD) stained separately with the individual antibodies used in the test samples. Flow cytometry data were analyzed using FlowJo (version 8.8.3; Tree Star, Inc.). For polychromatic analysis, initial gating for each sample set used a forward scatter area versus a forward scatter height plot to gate out cell aggregates. The cells were then gated through a forward scatter area versus a side scatter height plot to isolate small lymphocytes. A CD3 versus Vivid/Aqua plot was then used to remove dead cells. The cutoff point for positive CD4 T cell responses was 2-fold the negative control and at least 0.1% cytokine positive after subtraction of the background.
Virus growth and characterization.
CCR5-tropic HIV-1 BAL was grown on phytohemagglutinin (PHA; Sigma-Aldrich)-stimulated PBMCs in CM supplemented with IL-2 (Chiron). HIV p24 antigen in cell culture supernatants was monitored by ELISA (Beckman Coulter). Virus was harvested at the peak of infection, on either day 4 or 7. Viruses were concentrated by ultracentrifugation at 30,000 rpm for 70 min at 4°C, and virus pellets were resuspended in fresh RMPI at 10–50× to obtain clean, concentrated virus stocks. Control concentrated conditioned supernatant from uninfected PHA-stimulated T cells was collected as a control. The viral titers were determined by sensitive 14-d end-point titration assays using PHA- and IL-2–stimulated PBMC, and listed as 50% tissue culture infectious doses per ml (TCID50/ml), as described previously (Mascola et al., 1996). The final titer of the stock of HIV Bal was 2.6 × 106 TCID50/ml.
In vitro HIV infection of primary CD4 T cells.
Previously cryopreserved PBMCs were thawed for co-culture, washed thoroughly, and labeled with 0.25 µM CFSE fluorescent dye (Invitrogen). CFSE-labeled cells were washed thoroughly and resuspended at 2 × 106 cells/tube in CM and stimulated for 24 h with 1 µg/ml SEB at 37°C. Cells were then washed, resuspended in 200 µl of HIV-1 BAL stock (0.26 multiplicity of infection), and incubated at 37°C for 2 h. The residual virus was then washed off and cells were resuspended in 1 ml of CM with 1 µg/ml SEB, and then incubated at 37°C for an additional 22 h. 10 µg/ml BFA (Sigma-Aldrich) was added to all tubes during the last 4 h before immediate analysis via flow cytometry. Stimulation was performed in the presence or absence of 1 µM AZT or 10 µg/ml anti–IL-2 antibody (BD). In some experiments, CFSE was not used, and HIV infection was detected by staining for p24 antigen.
Cell sorting.
PBMCs from 20 HIV+ subjects with active TB were thawed, washed twice in the presence of 50 U/ml benzonase, and resuspended at 3 × 106 cells/ml in CM. Costimulatory antibodies (αCD28 and αCD49d; 1 µg/ml final concentration) were added to cells before splitting them into different tubes for overnight stimulation at 37°C with either MTB antigens or no antigen. BFA (10 µg/ml) was added to all tubes 2 h after stimulation. After incubation, cells were washed and stained with pretitrated surface antibodies as described in Stimulation and flow cytometric analysis. Cytokine-producing CD4 T cells were sorted with a modified FACSAria flow cytometer (BD) by gating tightly on live memory CD3+CD4+ T cells. MIP-1β+TNF+ memory CD4 T cells were then sorted and all other memory CD4 T cells were further delineated and sorted according to IFN-γ and IL-2 staining into IFNγ+IL-2+MIP-1β−, IFNγ+IL-2−MIP-1β−, and cytokine negative memory CD4 T cells. Between 193 and 50,000 fixed memory CD4 T cells from each of the four different populations were collected, depending on the number of PBMCs available and the response level for each individual. Cells were then stored at −80°C before gag DNA analysis.
Quantification of HIV gag viral DNA.
HIV DNA was quantified by qPCR with an ABI7700 (PerkinElmer) similar to a previously described protocol (Douek et al., 2002). HIV gag primers and probe were optimized for detection and quantification of East African subtype A and C strains. Gag primer position and sequences were as follows, 783gag, forward, 5′-GAGAGAGATGGGTGCGAGAGCGTC-3′ (Tm>60), 895gag, reverse, 5′-CTKTCCAGCTCCCTGCTTGCCCA-3′ (Tm>60); FAM-labeled probe 844gagPr, 5′-ATTHGBTTAAGGCCAGGGGGAARGAAAMAAT-3′. Sites where base sequences differed among HIV isolates were made degenerate and are noted as follows: where degenerate bases are denoted as R (A or G), M (A or C), K (G or T), H (A or C or T), and B (C, G, or T). To quantify the cell number in each reaction mix, the human albumin gene copy number was also assessed by qPCR. Albumin primer and probe sequences were as follows: hAlb, forward, 5′-TGCATGAGAAAACGCCAGTAA-3′; hAlb, reverse, 5′-ATGGTCGCCTGTTCACCAA-3′; and hAlbProbe, 5′-FAM-TGACAGAGTCACCAAATGCTGCACAGAA-BHQ1-3′. Sorted T cells were lysed in 0.1 mg/ml proteinase K buffered with Tris-Cl for 1 h at 56°C, and then for 10 min at 95°C to inactivate the enzyme. 5 µl of lysate was used in a total reaction volume of 25 µl containing 12.5 µM primers, 5 µM probe, a 10-mM concentration of each deoxynucleoside triphosphate, 3.5 mM MgCl2, 1.25 mM blue 636 reference dye, and 0.625 U platinum Taq in the supplied buffer. Reaction conditions included 5-min activation at 95°C, followed by 15 s at 95°C and 1 min at 60°C for 45 cycles. Quantification was generated using standard curves for HIV-1 gag and albumin. Cell-associated gag DNA from different T cell subsets from the same subjects were quantified during the same PCR run to assure comparability of the results.
Statistical analysis.
Data analyses were performed using Prism version 4.0 software (GraphPad, Inc.). Comparisons of two groups were performed using the Mann-Whitney test. Comparisons of paired groups were performed using the Wilcoxon matched pairs test. Tests used for statistical analysis are described in the figure legends.
We thank Mr. Weston Assisya for the coordination of field studies and Jutta Jung for coordinating TB diagnosis in Mbeya, Tanzania. We would also like to thank Drs. John Tetteh, Sampson F. Ofori, and Mike Ofori for donor recruitment and sample collection in Ghana. We also thank all donors for their contribution to the study.
This study has been supported by the European Commission through grants ICA_CT-1999-10007, ICA_CT-2002-10048, and E.C. DGVIII – AIDCO - SANTE/2006/129-931 and a Netherlands Organisation for Scientific Research WOTRO Grant (01.53.2004.025).
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MTB- and CMV-specific CD4 T cells are lost at different rates after HIV infection. (A) The frequency of MTB-specific (black, 5 PPD responding subjects, who remained TB asymptomatic) and CMV-specific memory CD4 T cell responses (gray; n = 6 CMV responding subjects who remained CMV disease free) at 6–12 mo after HIV seroconversion as the percentage of baseline response detected at 3 mo before the first HIV-seropositive follow up in latently infected subjects. The frequency of MTB- or CMV-specific CD4 T cell responses in chronically HIV-infected subjects (median time since HIV infection >3 yr; n = 17) and a HIV− control group (n = 17) is shown in B and C, respectively. The limit of detection is indicated. Memory CD4 T cells were defined by expression of CD27 and CD45RO. IFN-γ+ memory CD4 T cells were detected after in vitro stimulation of PBMCs with PPD or whole inactivated CMV virus by intracellular cytokine staining. Statistical analysis was performed using the Mann-Whitney test.
Differences in cellular maturation between MTB- and CMV-specific CD4 T cells are diminished during active TB disease and are associated with diametrically opposed production of IL-2 and MIP-1β. (A) Surface expression of the maturation markers CD27, CD45RO, and CD57 on total CD4 T cells (black) is shown for representative subjects as a density plot overlay. MTB-specific CD4 T cells in the absence (blue, HIV−) or presence (green, HIV+) of active TB disease and for CMV-specific CD4 T cells (red). The percentage of CD27+CD45RO+, CD27−CD45RO+, and CD57+ subsets within MTB- and CMV-specific CD4 T cells from HIV− subjects (blue, n = 17) and HIV+ subjects with active TB (green, n = 11; bottom). (B) Flow cytometric analysis of IFN-γ, IL-2, MIP-1β, and TNF production within pathogen-specific CD4 T cells. PBMCs from subjects with latent MTB infection (defined by positive response to region of difference 1 [RD1] antigens) or from HIV+ subjects with active TB were stimulated with MTB-antigens (PPD or a mix of PPD and RD1 peptide pools) or CMV whole antigen. The pie charts show the fraction of cells with 1, 2, 3, or 4 functions. The color-coded circles indicate the proportion of the 4, 3, 2, and 1 functional responses response that are contributed by the single cytokines IFN-γ (red), IL-2 (blue), Mip-1β (green), and TNF (pink). The fraction of cells that produce MIP-1β (right top) or IL-2 (bottom right) is shown as percentage of total cytokine-positive CD4 T cells. The median is indicated. Further delineation of the 16 different possible cytokine combinations is shown (bottom). (C) Intracellular staining of IL-2 (y axis) and MIP-1β (x axis) within CD27+CD45RO+, CD27−CD45RO+CD57−, and CD27−CD45RO+CD57+ CD4 T cells after stimulation with PPD or CMV is shown for one representative subject (left) and for all studied responses further delineated by IL-2 and MIP-1β production and presented as percentage of total cytokine-positive response (right). Statistical analysis was performed using the Mann-Whitney test (***, P < 0.0005; **, P < 0.005; *, P < 0.05).
HIV infection is characterized by increased fractions of MIP-1β+ and decreased fractions of IL-2+ CMV-specific CD4 T cells. (left) The fraction of IFN-γ+, TNF+, MIP-1β+, or IL-2+ among total cytokine positive CD4 T cells (y axis) is shown for HIV− (n = 11) and HIV+ subjects (n = 16). (right) The fraction of IL-2+MIP-1β+, IL-2+MIP-1β−, and IL-2−MIP-1β+ among total cytokine-positive CD4 T cells. PBMCs were stimulated overnight with whole inactivated CMV and the background (unstimulated control) was subtracted. Statistical analysis was performed using the Mann-Whitney test.
Productive HIV-1 infection of SEB-responding CD4 T cells is inhibited by in vitro neutralization of IL-2. (A) Productive HIV infection of CD4 T cells was analyzed after gating on CD4low T cells and staining for HIV-p24 (y axis). Shown are dot plots from samples stimulated in the presence or absence of 1 µM AZT. (B) Dot plots from samples stimulated in the presence (bottom) or absence (top) of a neutralizing anti–IL-2 antibody comparing CFSE-fluorescence intensity (left), surface CD25 (middle), and CCR5 (right) with HIV-p24 staining. PBMC samples were stimulated for 24 h with SEB and then infected for another 24 h with the CCR5 tropic HIV-1 strain BAL.
The capacity to secrete IL-2 by antigen-specific CD4 T cells is associated with increased CD25 expression. (A) Representative histogram of the CD25 expression on cytokine-negative, MIP-1β+IL-2−, and MIP-1β−IL-2+ CD4 T cells after 6 h of SEB stimulation of PBMCs. (B) The fraction of CD25+ CD4 T cells among the same subsets (n = 7). Statistical analysis was performed using the Mann-Whitney test.
In vitro HIV infection of cytokine-expressing cells. (A) Representative histograms of the HIV p24 staining from CD4 T cells delineated by intracellular staining for MIP-1β, IFN-γ, and IL-2 and (B) the fraction of p24+ CD4 T cells delineated by intracellular staining for MIP-1β, IFN-γ, and IL-2 from six independent experiments. PBMC samples were stimulated for 24h with SEB and then infected for another 24 h with the CCR5 tropic HIV-1 strain BAL. Productive HIV infection of CD4 T cells was analyzed after gating on CD4low T cells and staining for HIV-p24.
In vivo HIV gag DNA in MTB-specific CD4 T cells. (A) Gating/sorting strategy used to sort different memory CD4 T cell populations delineated by IFN-γ, IL-2, and MIP-1β production. MIP-1β− memory CD4 T cells were further delineated into IFNγ+IL-2+TNF+, IFNγ+IL-2−TNF+, and cytokine-negative memory CD4 T cells. The HIV gag DNA/10,000 cells determined within these populations is indicated. (B) The ratio of HIV gag copies/10,000 cells detected within cytokine-positive to cytokine-negative memory T cells from 16 cytokine-positive CD4 T cell populations sorted from 10 HIV+ subjects (different symbols) with active TB. (C) The mean number of gag copies/10,000 cells detected in MTB-specific CD4 T cells or total memory CD4 T cells is shown for each subject. Memory CD4 T cells were defined by expression of CD45RO and CD27. The PBMCs were stimulated overnight with a mix of PPD and RD1 peptide pools and further analyzed as described in the Materials and methods section. Gag DNA within different CD4 T cell populations of the same subject was quantified during the same RT-PCR run. The statistical analysis in C was performed using the Wilcoxon-rank-matched pairs test.
Characterization of Human CD8+ T Cells Reactive with Mycobacterium tuberculosis–infected Antigen-presenting Cells
Interleukin 27R regulates CD4+ T cell phenotype and impacts protective immunity during Mycobacterium tuberculosis infection
Induction of M3-Restricted Cytotoxic T Lymphocyte Responses by N-Formylated Peptides Derived from Mycobacterium tuberculosis


text:
Endiama and Al Rosa make official acquisition of 16.4 percent of Wargan in Sociedade Mineira de Catoca
Angola's National Diamond Company (Endiama) and Russian diamond company Al Rosa have formalized the acquisition of Wargan's 16.4 percent stake in Sociedade Mineira de Catoca, both raising its stake to 41 percent, it was announced last Friday.
According to a note from the Angolan diamond company, with the cessation of 16.4 percent of shares in Wargan, Endiama and Al Rosa will have 41 percent stakes in Sociedade Mineira de Catoca, with LL International maintaining the its 18 percent stake.
The process of acquiring Wargan's shares was announced on January 21 of this year by the administrator for Geology and Mining Development at Endiama, Ana Feijó.
At the time, Ana Feijó said that around 70 million dollars were paid to Wargan, with the company's equity.
"The main objective of these actions is to reinforce the role of Endiama, as an operating company. We will no longer be concessionaires and will become a company that will not only manage shareholdings, but assume its fundamental role, its core business', which is prospecting, production, ore treatment, cutting and marketing", she highlighted.
Ana Feijó stressed that these actions will promote increased production, greater profitability, increased income and, consequently, the improvement of workers' lives.
Three years ago, Lusa reported that the President, João Lourenço, signed the deal to sell Odebrecht's 16.4 percent stake in Brazil's corruption scandal in Sociedade Mineira de Catoca, which operates the fourth-largest diamond mine the open air of the world.
The deal had been agreed by mine partners in eastern Angola in August 2017 and was enacted by presidential decree on January 4, 2018.
The document stated that the participation of the Brazilian group, through its branch in Germany, Odebrecht Mining Services Investments GmbH, was sold in favor of the company Wargan Holdings, which in turn is 100 percent owned by the Russians of Al Rosa PJSC, operating in Catoca.
The decree further defined that Wargan Holding would dispose of the same 16.4 percent stake to Al Rosa and Endiama, "in accordance with the specific terms of commitment agreed to that effect" in equal shares of 8.2 percent.
With the deal, Sociedade Mineira de Catoca now has a shareholder structure led by Al Rosa and Endiama, both with a 41 percent stake, while the Chinese of LL International Holding BV will maintain 18 percent.
In the presidential decree of January 4, 2018, it was stated that Odebrecht Angola "completed fully the defined purpose of taking the Sociedade Mineira de Catoca Limitada together with Endiama EP and the other shareholders to operational maturity", but that it "has manifested the intention to divest its share" to "focus on its infrastructure projects".
Angola raised 1.95 billion dollars from the sale of diamonds in 2022
Angola holds this month's first International Diamond Conference to attract investment

redpajama_set_name: RedPajamaCommonCrawl


text:
Who is really to blame for the failure of the Syrian revolution?
Basheer Nafi
Published date: 30 April 2018 13:03 UTC | Last update: 2 years 8 months ago
The Arab revolutionary movement did not find powerful international allies to support and protect it at key moments of change
Launched with the call for reform, the Syrian revolution soon progressed into demanding the regime's downfall. But neither through peaceful popular mobilisation nor through the use of force have Syrians been able to bring down President Bashar al-Assad.
Since 2011, the Syrian revolution has turned into a bloody civil war and an arena for regional and international conflict. Despite the massive physical destruction, hundreds of thousands of dead and millions of refugees, Assad continues to represent Syria in the international arena, administering the affairs of what remains of its state institutions.
With considerable support from its Russian and Iranian allies, the regime has made steady progress in its war against the armed factions of the Syrian revolution.
Opposition in disarray
Since failures always prompt a search for those responsible, discussions about the demise of the revolution have taken off, and not just within Syrian revolution circles. In one view, the Syrian opposition, both its military and political elements, is principally responsible.
The political opposition has not been able to unite its ranks or introduce a charismatic leadership around which the people could rally, to convince the world of its seriousness in representing the revolution and the people.
Western support for the Arab revolutions and for the process of democratic transformation was slow and reluctant. It soon turned into indifference, or a return to the policy of "the devil we know".
In addition to the huge gap separating the revolution's political and military wings, there are dozens of military factions that vary in size.
When it became necessary for armed opposition forces to protect the revolution's integrity, they refrained from confronting al-Qaeda and the Islamic State (IS) and failed to expel them from the Syrian arena. Most of these armed groups appeared more anxious to preserve a paper entity than to safeguard the interests of the people and the revolution.
This has not been an opposition qualified, at any given moment, to lead the people or to meet the challenges imposed by the transformation of Syria into a battleground. As soon as the balance of power started to shift in favour of the regime, the opposition was unable to remain steadfast.
Broader dynamic for change
Although largely accurate, this condemnation of the opposition implies another condemnation of the Syrian people. After all, they are the ones who gave rise to this opposition. This logic leads to the conclusion that the Syrian people were not ready for the confrontation with Assad's oppressive regime, and that the victorious party is more qualified to lead Syria.
What this viewpoint overlooks is that the Syrian revolution was not an isolated phenomenon. It emerged within a wider context of an Arab revolutionary dynamic for change. It would be wrong to read what occurred in Syria in isolation.
Syrian boys play with a basketball as a government soldier walks by in the town of Hazzeh in Eastern Ghouta on 28 March 2018 (AFP)
Today, seven years after the eruption of the revolutions in Tunisia, Egypt, Libya, Syria and Yemen, it can be said that the objective of changing the political order did not fail in Syria alone, but in every single one of these states.
The failure occurred in countries that did not witness a resort to arms, did not see the emergence of terrorist groups such as IS and al-Qaeda, and contained more mature opposition forces.
Even when the revolutions succeeded in bringing down regimes, the old ruling classes soon bounced back, launched counterrevolutionary movements and regained power.
The balance of power quickly turned against the Arab revolutionary movements in all these states. This reversal did not occur due to the naivete of the opposition or its incompetence, but due to the crystallisation of new compelling circumstances that overwhelmed the opposition and the people.
Counter-mobilisation
The fact that the movement of revolution and change was pan-Arab, and not just Syrian or Yemeni or Libyan, triggered a counter-mobilisation across the Arab world. As soon as the forces of the counterrevolution realised the magnitude and impact of the revolutionary dynamic, it moved to build a wide-ranging coalition.
Even without prior agreement, influential states with enormous political, military and financial resources - such as Saudi Arabia, the United Arab Emirates and Iran - moved to bring about the defeat of the movement of change and democratic transformation.
The counterrevolution camp considered the democratic revolutions as a threat to its privileges, powers and influence within the Middle East, and thus decided to wage war across the entire region.
Even if he 'wins', Assad has lost Syria
Sami Hamdi
The Arab revolutionary movement, furthermore, did not find powerful international allies to support and protect it at key moments of change. In an entwined world, no democratic transformation has ever succeeded without external backing. Without US and European political and economic support, no democratic change would have established roots in Spain and Portugal in the aftermath of the collapse of their despotic regimes, nor in Eastern Europe in the aftermath of the collapse of the communist bloc.
Relinquishing power or waging war
There has been something specific about Syria. In all cases of political change during the past century, ruling classes have had to choose, upon facing wide-scale popular opposition, between two main paths: relinquishing power or waging a bloody war against the people. Despots such as the Shah of Iran, Zine El Abidine Ben Ali, Hosni Mubarak and Ali Abdullah Saleh relinquished power, although with some reluctance.
In an extremely complex historical path of transformation, there is no such thing as the end of the road
Despots such as Muammar Gaddafi and Assad chose confrontation. Gaddafi was brought down by a decisive foreign intervention, while Assad barricaded himself behind a bloody form of sectarianism. As soon as he discovered his inability to achieve victory against the revolution and the people, he promptly invited regional sectarian support. When he and his regional allies failed, he did not hesitate to appeal to the Russians, even at the cost of losing his sovereignty.
It is true that the Arab revolution failed to achieve its objectives of change and democratic transition. However, the causes of this failure were much bigger than the shortcomings of the people's movement and opposition forces. At the same time, what should be remembered is that the current failure is not the end of the road. In an extremely complex historical path of transformation, there is no such thing as the end of the road.
- Basheer Nafi is a historian of Islam and the Middle East.
The views expressed in this article belong to the author and do not necessarily reflect the editorial policy of Middle East Eye.
Photo: A Syrian government soldier walks past destroyed buildings in the former rebel-held town of Jobar in Eastern Ghouta on 1 April 2018 (AFP)
In pictures: Floods devastate camps in Syria's Idlib
Heavy rainfall floods Syrian refugee camps in Idlib
Pompeo's dinner with Mossad was about egos, not Syria strikes
Syria's war is far from over
Christopher Phillips
I'm Syrian, and I will not give up hope for justice
Razan Saffour
Assad's land grab: Refugees face losing the homes they fled under new law


Extraordinary Ministers of the Eucharist bring the Eucharist to sick and/or homebound parishioners weekly and at Brighton Gardens Sunrise Facility on Tuckerman Lane. They prepare a monthly Mass and weekly Rosary at Brighton Gardens where they assist residents.
Interested volunteers may contact Charlotte King. Requires initial one-time training and about one hour a week to distribute Communion. Requires reverence for the Eucharist; ease in public settings; Catholic in good standing who has received the Sacraments of Initiation (Baptism, Confirmation, and Eucharist), being commissioned as an Extraordinary Minister of Communion after attending a special class on the Eucharist.


Jui's Chaupati is the only place which provides delicious foods such as paratha, south indian, sandwiches, pav bhaji, missal, snacks, chaat, pulav and other breakfast foods. We are also providing cold drinks, ice cream, juices, shakes & mastani etc. We are providing these services from last 15 years. We are qualified ISO 9001-2008 registered restaurant so we are provide only quality foods all the time. Our motto is providing hygienic best foods & pure veg. Our both branches is located at prime location of pune city i.e Khardai & Wagholi. We are providing home delivery services for kharadi, wagholi & viman nagar area.


text:
Officials: Shop owner tracked down, smashed thief's hands with hammer
by: James Selby
BRIDGE CITY, La. (KLFY) — (12/11/19) Authorities say the owner of a Bridge City auto shop owner kidnapped a man he suspected of stealing tools from his business, beat the man and smashed both of his hands with a hammer.
The Jefferson Parish Sheriff's Office says 37-year-old Chad Baxter saw someone on surveillance video last month who was stealing from his shop. Officials say Baxter tracked down 39-year-old Dorian Lewis on Thursday. The man says Baxter forced him into a vehicle at gunpoint.
Officials say Baxter allegedly beat Lewis in the back of his auto shop. Both men have been arrested.
It's unclear whether they had attorneys who could speak for them.
Stay up to date with the latest news by downloading the KTVE/KARD News App from the App Store or Google Play.
Italy (AP) (01/20/21)-- Italy's virus czar is pressing ahead with plans to take legal action against Pfizer after the U.S. pharmaceutical company announced delays in delivering pre-ordered COVID-19 vaccines to Europe.


text:
from google.cloud import gaming_v1beta

def sample_create_game_server_config():
    # Create a client
    client = gaming_v1beta.GameServerConfigsServiceClient()

    # Initialize request argument(s)
    request = gaming_v1beta.CreateGameServerConfigRequest(
        parent="parent_value",
        config_id="config_id_value",
    )

    # Make the request
    operation = client.create_game_server_config(request=request)

    print("Waiting for operation to complete...")

    response = operation.result()

    # Handle the response
    print(response)

# [END gameservices_v1beta_generated_GameServerConfigsService_CreateGameServerConfig_sync]

redpajama_set_name: RedPajamaGithub


he explained. Sunlight poured through gaping holes in the roof, and the coarse brown wrapping paper pasted on the walls was splattered and streaked by rain. The open door of Cordelia's bedroom revealed a wooden bed, a marble-topped bureau, and a washstand of the Victorian period. A rocker, two straight chairs, a small table, and a trunk completed the furnishings of the room and left but little space for its occupant to move about.


text:
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Author: Geek's Guide to the Galaxy. Geek's Guide to the Galaxy Culture
Date of Publication: 08.04.18. 08.04.18
Time of Publication: 8:50 am. 8:50 am
American Horror Story: Cult brought US politics into the show. FX Networks
The horror genre is full of books and movies that involve politics or that make a political statement, including Dawn of the Dead, They Live, and The Dead Zone. This affinity between horror and politics comes as no surprise to political commentator Ana Marie Cox.
"Horror is almost inherently political," Cox says in Episode 320 of the Geek's Guide to the Galaxy podcast. "It's one of the reasons I love it. Tell me what you're afraid of and I'll tell you who you are."
https://www.podtrac.com/pts/redirect.mp3/https://www.wired.com/wp-content/uploads/2018/08/geeksguide320final.mp3
Those parallels have become even more apparent in the current climate thanks to shows like American Horror Story: Cult, which uses footage of President Trump's election night victory to unsettle viewers. That move, though, was one fantasy author Sam J. Miller thinks was a step too far.
"I thought the political-ness of it was so hamfisted and so on-the-nose that it wasn't doing anything interesting or new," he says. "It was just kind of reveling in the horror of our moment and rubbing our noses in it, as if we didn't get enough of that every day."
Cox also notes that we're starting to see more real-life connections between the worlds of horror and politics. "The person who was the Blumhouse publicity person wound up going to work for Jared Kushner," she says. "He left recently, and when the White House becomes too much of a shit show even for someone who's used to promoting The Purge, maybe there's a problem."
Horror author Grady Hendrix feels that today's horror writers have some catching up to do, given the extent to which real-life terrors have outpaced fictional ones.
"I feel like horror has really not engaged in a direct way with the changing vocabulary of the world around us," he says. "To me, these days the language of horror is swatting, and black sites, and false flag operations, and and MKUltra and all this. And you don't see that a lot in horror."
Listen to the complete interview with Ana Marie Cox, Sam J. Miller, and Grady Hendrix in Episode 320 of Geek's Guide to the Galaxy (above). And check out some highlights from the discussion below.
Sam J. Miller on zombies:
"The thing that I love about zombies is I feel like they're the only real monster of the major movie monster categories that doesn't come from Western folklore or a Western author's imagination. … Zombies are a Haitian folklore creature that sort of represents a re-imagining of slavery—this image of somebody's free will being lost, and being commanded by another. To me I think the proper context for Night of the Living Dead isn't so much Vietnam—although that's a factor—as the civil rights movement. What these movies often are about is the end of somebody's world, and usually it's the end of white supremacy. It's the idea that suddenly people are fighting back and everything has been called into question."
Grady Hendrix on The Purge:
"The Purge is basically the same story as Shirley Jackson's 'The Lottery.' It's this idea that for society to be healthy, they have to perform a sacrifice. That's The Wicker Man. That's a really old horror trope, that people have to get their bloodlust out, and then everybody's really happy, and the corn is thick and ripe, and we'll have a great harvest, and there'll be no crime, and unemployment will drop way down. I mean, what's the difference between low unemployment numbers and a good corn harvest? It's a little temporal shifting. I agree that the Purge movies aren't as smart as they think they are, but they're really, really interesting."
Geek's Guide to the Galaxy
The Polity Is Libertarian Space Opera Done Right
The Ada Lace Books Will Get Girls Interested in STEM
Satanists Just Aren't Scary Anymore
Ana Marie Cox on right-wing horror:
"I have a weird fascination with right-wing figures who try to write fiction about the stuff that they believe. Like I've read all of Glenn Beck's novels, which are all basically just dramatizations of what he actually believes. They're terrible, and it's because he's just writing down a slightly more elaborate version of the stuff that you can hear on conservative, right-wing talk radio all the time. You know, the United Nations is taking us over, Agenda 21—which sounds like it's supposed to be about golf courses, but really is about FEMA concentration camps. It makes terrible horror. He's like, 'I find this idea of the United Nations taking over the US very scary, so I'm going to write a book that shows people how scary it is.' But it's not scary."
David Barr Kirtley on The Dead Zone:
"One thing that did actually have a really big influence on me was watching the movie of Stephen King's The Dead Zone. There's this scene where the president—who's Martin Sheen—they bring the nuclear button in to him, and he gives this big crazy religious/apocalyptic speech and presses the button, initiating worldwide nuclear war, moments before his aides run in and say, 'It's not necessary. We've worked out a peace settlement.' And that has always just been very, very haunting to me, and has always stuck in my mind so much, and it did really—at least for me—make me much less likely to support a candidate who I could imagine in a scene like that."
This fake conspiracy site is fully post-parody
How to free up space on your iPhone
Inside the moonshot factory building the next Google
The challenge of teaching helicopters to fly themselves
The political education of Silicon Valley
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'Immer, Zlaz' Reveals the Private Life of a Sci-Fi Genius
'Andor' Is a Master Class in Good Writing
Here's How Robert Jordan Built 'The Wheel of Time'
'Control Freak' and the Power of the Intimate Video Game Memoir
'A Fire Upon the Deep' Is Mind-Blowing Space Opera
New Mexico Is a Great Place for Sci-Fi
'Poster Girl' Explores the Surveillance State's Allure
'A Canticle for Leibowitz' Is a Big, Beautiful Mess
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text:
require File.join(File.dirname(__FILE__), '..', 'notifier')

module AuthHelpers
  module Model

    # Adds a module that deals with forgot your password. It overwrites the
    # reset password method from authlogic for one that accepts a password.
    #
    module Recoverable
      def self.included(base)
        base.extend ClassMethods
      end

      def reset_password(new_password, new_password_confirmation)
        self.password              = new_password || ""
        self.password_confirmation = new_password_confirmation || "" if self.respond_to?(:password_confirmation)
      end

      # Reset the password with the new_password is equals its confirmation and
      # set reset password code to nil.
      #
      def reset_password!(new_password, new_password_confirmation)
        reset_password(new_password, new_password_confirmation)
        self.save
      end

      module ClassMethods

        # Receives a hash with email and tries to find a record to resend reset
        # password instructions. If the record can't be found, it sets the
        # appropriate error messages and return the object.
        #
        def find_and_send_reset_password_instructions(options={})
          recoverable = AuthHelpers.find_or_initialize_by_unless_blank(self, :email, options[:email])

          if recoverable.new_record?
            recoverable.errors.add(:email, :not_found, options)
          else
            recoverable.reset_perishable_token!
            AuthHelpers::Notifier.deliver_reset_password(recoverable)
          end

          return recoverable
        end

        # Receives a hash with perishable_token, password and password confirmation.
        # If the password cannot be reset (confirmation fails, for example), it
        # returns an object with errors.
        #
        def find_and_reset_password(options={})
          if recoverable = self.find_using_perishable_token(options[:perishable_token])
            recoverable.reset_password!(options[:password], options[:password_confirmation])
            recoverable
          else
            AuthHelpers.new_with_perishable_token_error(self, :invalid_reset_password, options)
          end
        end

      end

    end
  end
end


The following is an excerpt from the book Purpee the Purple Dragon and the Stone-Eyed Snake. For every two Purpee books sold on Amazon.com, we donate one to a children's hospital. For details click here.
Stinky replied, "I don't know but I don't want to find out." At the same time, Purpee said excitedly, "Let's go find the snake and see if it is true!" All of them jumped up and down in agreement except for Stinky who was nervous about looking for the snake. They packed a backpack with some search tools and snacks and headed on their way to the woods behind the park.
Purpee, Shelly, and Stinky took the trail on the left, while Sunny, Smoky, and Pepper followed the trail on the right.
Purpee got down on his hands and knees to check out the hole but it was too dark and he couldn't see anything. "Stinky, hand me the flashlight in the backpack." Once again, Purpee crawled into the hole as far as he could fit and turned the flashlight on, looking for the snake. All of a sudden, he heard a growl.
Please consider purchasing the book on Amazon.com. For every two Purpee books sold on Amazon.com, we donate one to a children's hospital. For details click here.


When selecting a topic for your research paper, you might experience some difficulties. To make this process easier, we have come up with the list of steps you should take. Using these steps, you will certainly choose the best topic for research paper and will write it successfully!
It is crucial to know what your teacher is expecting from you. For that, you need to understand what area you have to research, what the deadline is, how the paper should be formatted and how long it should be. It's also important to know in advance what kind of resources you can utilize and how many of them should be there.
Now, you're ready to start thinking about the topic. You can do it by brainstorming a bunch of various ideas. For example, if you need to write about modern technologies, you can think about writing a paper on their influence on people and relationships. If you have to write about art, you can choose the art of a particular era. Brainstorm your ideas by making lists and evaluating them or by creating a mind map.
When you're done brainstorming and have some topic ideas, you can start narrowing them down. You should do this by finding reliable resources, because it's not a good idea to choose a topic for which you can find no relevant information. Remember to make your topic as narrow as possible. If your topic is too broad, you might have a lot of problems, such as trouble concentrating on certain points to prove your thesis. It's better to stay away from hotly debatable social topics, such as gun control, racial issues, abortion, religion, etc (unless the assignment requires that, of course). Think about the topics where your reader can learn something new.
If choosing a topic remains an issue, discuss it with your friends, family members, peers or teachers. Looking at your situation from aside, any of these people can offer you the advice you need to finally make a good choice.
Now the time has come to ask a question about your topic and come up with the thesis that you are going to prove or disprove with your research. If you formulate a good thesis, you will certainly do a good research. Remember to support your argument while researching and writing. Don't drift off the topic.
If you follow these simple steps, choosing a topic for your research paper will become much easier. When you do choose a good and interesting topic, all you will need to do is to remain focused, research it continuously and enjoy writing your research paper!


Be the first to share your favorite memory, photo or story of Seth. This memorial page is dedicated for family, friends and future generations to celebrate the life of their loved one.
Seth Michael Chemacki, Age 40 of Deerfield Beach, FL, formerly of Grand Ledge, MI, passed away on Wednesday, January 30, 2019 at Broward Health North in Deerfield Beach. He was born on October 8, 1978 in Lansing, Michigan, to Michael Victor and Sylvia Kay (Olmstead) Chemacki.
Seth was an amazing Husband, Son, Brother, Nephew, Uncle and friend. Everyone who knew him became family. Seth enjoyed spending time with family and friends fishing, camping, watching football or just "crusin". Whether in his big, green truck or in his fast, blue Corvette, you were along for the ride when with him. He had a love for his profession as an Electrical Engineer, as well as a love for anything and everything electronic.
He is survived by his wife of 14 years, Antoinette "Tonie" Chemacki, Mother Sylvia Kay Chemacki, Brothers; Elton Joseph Chemacki and Jordan Lee (Lindsey Edmiston) Chemacki, In-Laws: Ralph and Sami Picariello, Brother-in -Law, Tony (Jennifer), Nieces and Nephews; Aubrey Edmiston, Adelyn Chemacki, Tyson Chemacki, Amanda Picariello, Savanna Picariello, Jaycee Picariello, and Great Niece, Delilah Picariello. He is preceded in death by his Father, Michael Victor Chemacki in 2016.
To send flowers or a remembrance gift to the family of Seth Michael Chemacki, please visit our Tribute Store.
"Email Address" would like to share the life celebration of Seth Michael Chemacki. Click on the "link" to go to share a favorite memory or leave a condolence message for the family.


Asensible path to declare New York law starts with these two steps: (1) if the duration is definite, the at-will doctrine is inapplicable, on the other hand, (2) if the employment term is indefinite or undefined, the rebuttable at-will presumption is operative and other factors come into the equation….
When an agreement is silent as to duration, however, it is presumptively at-will, absent an express or implied limitation on an employer's otherwise unfettered ability to discharge an employee. Only when we discern no term of some definiteness or no express limitation does the analysis switch over to the rebuttable presumption line of cases. They embody the principle that an employment relationship is terminable upon even the whim of either the employer or the employee. The agreement in this case is not silent and manifestly provides a sufficiently limiting framework….
The range of the employment relationship, concededly created and actualized for several years in the framework of this Federal dispute, is established by the definable commencement and conclusion of Tyson's professional boxing career. Though the times are not precisely predictable and calculable to dates certain, they are legally and experientially limited and ascertainable by objective benchmarks.


The Niagara Falls Courtyard by Marriott Hotel is located in the Tourist District only one block away from the majestic Falls. Both Clifton Hill and Casino Niagara are located only a short distance from the hotel.
This 10-storey 258 guestroom hotel offers wonderful Niagara Falls accommodations for all types of travelers. With a Keg Steakhouse & Bar located in the hotel lobby and a T.G.I. Fridays Restaurant next to the hotel, all guests are provided with great dining options. Hotel amenities include an Indoor Pool & Outdoor Pool with waterslide and sundeck, a Universal gym with fitness equipment, Whirlpool and Sauna, Games Room and a Gift Shop.
The Niagara Falls Courtyard Hotel by Marriott is located in Niagara Falls, Ontario, Canada, in the heart of the tourist district and just one block from the world famous Niagara Falls. By staying at this hotel, guests are only a short distance from the Fallsview Casino Resort, Casino Niagara, Clifton Hill – the Street of Fun in Niagara Falls, and other famous Niagara Falls Attractions. Some attractions located within steps of the hotel include Dino Island 4-D Movie and Screamers Haunted House.
Within the hotel lobby level, you'll find the Courtyard Cafe, famous for its great breakfast buffet and made-to-order omelet station. Also within the hotel is the very popular Keg Steakhouse & Bar and next door is T.G.I. Fridays Restaurant for a great family dining experience.
The Courtyard by Marriott is a 10 storey hotel that has 258 guestrooms including luxurious whirlpool, fireplace, presidential and 2-room family suites. Guests will find all of the conveniences of home here including: high speed internet access, in-room coffee maker, mini bar, iron/ironing board, and hair dryer. Additional amenities include: casino shuttle, currency exchange, indoor and outdoor pools, gym, games room gift shop, on-site parking and additional shops, restaurants, and attractions within walking distance.
The Courtyard Cafe located on-site serves up a superb breakfast buffet including made-to-order omelets. The Keg Steakhouse & Bar is also located on-site and is open for lunch and dinner and is famous for its steaks. TGI Friday's is an exciting dining experience with fabulous food. Try the mouthwatering Jack Daniel's Grill menu – steak, salmon, chicken or ribs basted in a sweet-smokey mountain J.D. sauce. Open for Lunch and dinner.
The Courtyard by Marriott Niagara Falls is the perfect place to hold your next meeting. Amenities include: 3000 square feet of meeting facilities, in house audio visual services, high speed interent and 2 meeting rooms. Located in the heart of the tourist district, the Courtyard by Marriott Niagara Falls is close to the Falls and Casinos, as well as a host of attractions that make Niagara famous! Our professional meeting planners will assist in making sure your next Niagara conference or event is a success!


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Q: How to disable Google Chrome (or Chromium) start page ads / promotions? When opening a start page in chrome, at the bottom-middle location there is this promotion from Google to "Use Family Link" and "Start your school year with a Google course". I don't have kids neither do I go to school.
I tried looking for available options in chrome://flags but couldn't find anythign that would look like it could disable this.
How can I disable these advertisements? My adblocker won't work on my start page unfortunately (it's disabled, probably for "security" reasons).
I tried downloading Chromium (sync) but it's still there.

A: If you are so annoyed because of it(like me), you can open settings > Go to On Startup > Select Open a specific page or set of pages and set google.com or whatever you prefer.
But yes this way you cant access the shortcuts.

redpajama_set_name: RedPajamaStackExchange


text:
namespace PivotalTrackerDotNet
{
    public interface IPivotalTrackerClient
    {
        IAuthenticationService Authentication { get; }

        IAccountService Account { get; }

        IStoryService Stories { get; }

        IMembershipService Memberships { get; }

        IProjectService Projects { get; }
    }
}


M2 is here from London for a visit. We were housemates there. We had a great time of shopping, chatting, eating, having ice cream, sitting in parks, museums and so on. Being together again reminded us both of what a great time we had in our house and how lucky we were to have housemates that were more like family. In a text message received today, she said: "I feel like in the old days when we lived together in peace and harmony. :)" Yes, me too. And it left me both yearning and rejuvenated.
And every few months, one of us writes an email, reliving those precious moments, once again taking us all through memory lane wrapped in bubbles of nostalgia :D Its the best and saddest feeling.
azra, it feels so good to be so understood! ;p yeah it is the worst good feeling in the world!