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|Publication number||US7357930 B1|
|Application number||US 09/980,516|
|Publication date||Apr 15, 2008|
|Filing date||May 3, 2000|
|Priority date||May 3, 1999|
|Also published as||CA2270600A1, CA2369550A1, EP1173220A2, WO2000066173A2, WO2000066173A3|
|Publication number||09980516, 980516, PCT/2000/469, PCT/CA/0/000469, PCT/CA/0/00469, PCT/CA/2000/000469, PCT/CA/2000/00469, PCT/CA0/000469, PCT/CA0/00469, PCT/CA0000469, PCT/CA000469, PCT/CA2000/000469, PCT/CA2000/00469, PCT/CA2000000469, PCT/CA200000469, US 7357930 B1, US 7357930B1, US-B1-7357930, US7357930 B1, US7357930B1|
|Inventors||Michel G. Bergeron, Andre Desormeaux, Michel J. Tremblay|
|Original Assignee||Infectio Recherche Inc.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (11), Non-Patent Citations (27), Referenced by (1), Classifications (22), Legal Events (2)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application claims priority to International Application No. PCT/CA00/00469, filed May 3, 2000 (Publ. No. WO0066173), and Canadian Application No. 2,270,600, filed May 3, 1999.
This invention relates to formulations of ligands capable of binding to host membrane proteins present at the membrane surface of infectious agents as well as at the membrane surface of the host cells. More specifically, this invention relates to ligand-conjugated vesicles that may be administered alone or in combination with a drug. The drug may be incorporated into the vesicles or administered concurrently. Even more specifically, the protein is HLA-DR or CD4, the ligand is an anti-HLA-DR or anti-CD4 antibody or antibody fragment and the vesicles are liposomes. The drugs are preferably directed to a disease caused by HIV. These formulations could be used either as a therapeutic or a preventive tool.
It is now well-established that in the early-stage of human immunodeficiency virus (HIV) infection and throughout the clinical latent stage, viral particles accumulate and replicate actively in lymphoid organs despite a low viral load in peripheral blood. The high viral load observed in the lymphoid tissues was reported to be partly associated with trapped HIV particles on the follicular dendritic cells (FDC) located in the germinal centers. In addition to the extracellular localization of HIV in interdendritic spaces of germinal centers, viral particles are also found within the endosomal and cytoplasm compartments of FDC. Moreover, viral particles bound to the FDC remained highly infectious to CD4+ T-cells despite the presence of neutralizing antibodies on their surface. Over the course of HIV infection, the FDC network was shown to be gradually disrupted and ultimately destroyed. The incapacity of FDC to retain HIV particles in advanced stages of the disease has been postulated to contribute to the increased viral burden in the periphery. As the microenvironment of lymphoid tissues is crucial for effective immune responses, it is important to reduce or abrogate the production and the accumulation of HIV-1 particles in these tissues to preserve both their architecture and integrity.
Highly active antiretroviral therapy (HAART), usually consisting in the combination of two nucleoside analogues and one protease inhibitor, has been shown to be effective to reduce the plasma viral load to undetectable levels in HIV-infected individuals. However, anti-HIV regimens do not fully eliminate viral replication in secondary lymphoid tissues and this continued replication of HIV-1 seems to be due to the presence of drug-sensitive viruses. In addition, replication-competent HIV-1 are routinely isolated from resting CD4+ T-cells from patients receiving HAART even after 30 months of therapy (Finzi et al., 1997, Science, 278:1295-1300). In fact, it was estimated that it would take as much as 60 years to eradicate HIV from an infected individual (Finzi et al., 1999, Nature Med. 5:609-611). In addition, it was shown that initiation of HAART in HIV-infected patients, as early as 10 days after the onset of symptoms of primary HIV-1 infection, did not prevent generation of latently infected resting CD4+ T-cells carrying integrated HIV-1 DNA despite the successful control of plasma viremia (Chun et al., 1998; Proc. Natl. Acad. Sci. USA 95:8869-8873). On the other hand, an increasing number of treatment failures resulting from toxicity, drug-resistant mutants and/or poor compliance of patients to drug regimen are emerging with long-term therapy.
All approved anti-HIV drugs are generally aimed at blocking viral replication within cells by inhibiting either HIV-1 reverse transcriptase or protease which are essential enzymes for viral replication. Antiviral drugs include anti-HIV drugs such as 3′-azido-3′-deoxythymidine (AZT, zidovudine), 2′-3′-dideoxyinosine (ddI; didanosine), 2′-3′-dideoxycytidine (ddC; zalcitabine), 2′-deoxy-3′-thiacytidine (3TC; lamivudine), indinavir, saquinavir, ritonavir, nelfinavir, ganciclovir, foscarnet and ribavirin. The development of new drugs that targets other events of HIV-1 infection or the use of alternative approaches for the treatment of HIV-1 infection remains a high priority to cure this deadly disease. Most polyene macrolide antibiotics are active against a variety of lipid-enveloped RNA and DNA viruses. The binding of these drugs to sterols located within microbial cell membranes modifies the permeability and function of cells decreasing the infectivity of lipid-enveloped virus. Both amphotericin B (AmB) and nystatin A have been shown to be efficient to inhibit the in vitro replication of HIV-1, probably via the binding of drugs to cholesterol in the membrane of HIV-1, which has a high cholesterol-to-phospholipid ratio. Antiviral activity of a series of more soluble derivatives of AmB has also been investigated. The mechanism of action by which these molecules inhibit HIV-1 infection was shown to be markedly different upon the nature of the AmB derivative, their main target being the viral and/or cellular membrane.
One common feature of retroviruses, as well as of many other enveloped viruses, is the acquisition of host cell surface molecules during the budding process. FDC, B lymphocytes, antigen presenting cells like macrophages and activated CD4+ T-cells are abundant in lymphoid tissues and all express substantial levels of the HLA-DR determinant of the major histocompatibility complex class II (MHC-II). Monocyte-derived macrophages, which are also CD4+ and express HLA-DR, are considered to be the most frequently identified hosts of HIV-1 in tissues of infected individuals. Given that HIV-1 has been reported to incorporate a vast array of cell membrane derived structures while budding out of the infected cell, the probability that newly formed viral entities will bear cellular HLA-DR is thus high. It has been demonstrated that plasma HIV-1 isolates from virally-infected individuals do carry on their surface host-encoded HLA-DR (Saarioos et al., 1997, J. Virol. 71:1640-1643). The physiological relevance of cellular HLA-DR bound to HIV-1 is further provided by previous studies from our laboratory indicating that HLA-DR is one of the most abundant host-derived molecules carried by HIV-1 (Cantin et al., 1997, J. Virol. 71:1922-1930). Numerous reports have provided evidence for the presence of many other host-derived components in different laboratory and/or primary isolates of HIV-1 (Table 1; for review see Tremblay et al., 1998, Immunol. Today 19:346-351).
There is serious doubt about the ability of current antiviral treatment to eradicate HIV-1 infection. Therefore, more potent antiretroviral drugs or the use of alternative approaches that block the potential spread of virus within lymphoid tissues, their main reservoir, are urgently needed. Considering that HIV accumulates and replicates actively within lymphoid tissues, any strategy that will decrease viral stores in these tissues might be beneficial to the infected host. As liposomes are naturally taken up by cells of the mononuclear phagocytic system (MPS), liposome-based therapy represents a convenient approach to improve the delivery of anti-HIV agents within lymphoid tissues. As host-derived HLA-DR proteins are abundantly expressed on antigen presenting cells such as monocyte/macrophages and FDC, liposomes bearing surface-attached anti-HLA-DR antibodies (anti-HLA-DR immunoliposomes) and containing anti-HIV agents constitute a convenient approach to target even more specifically HIV-1 reservoirs. Similarly, the coupling of anti-CD4 molecules on the surface of liposomes (anti-CD4 immunoliposomes) should lead to a specific targeting of both CD4 T-lymphocytes and monocyte/macrophages which represent the major cellular reservoir for HIV-1. Furthermore, as host-encoded HLA-DR determinant is also present on the virion's surface, incorporation of neutralizing agents, such as amphotericin B, in anti-HLA-DR immunoliposomes represents an attractive strategy to destroy cell-free viruses. Taken together, the use of immunoliposomal drugs represents an attractive strategy to target primary cellular reservoirs of HIV-1. Such a targeted drug delivery system could hopefully improve the treatment of HIV infection.
U.S. Pat. No. 5,773,027 discloses the use of antiviral agents encapsulated into liposomes for the treatment of viral diseases. However, this publication does not specifically teach liposomes bearing surface-attached antibodies comprising or not a drug capable of inhibiting replication or destroying both cell-free HIV virions and virally-infected cells. Such immunoliposomes could represent a novel therapeutic strategy to treat more efficiently this retroviral disease.
TABLE 1 Host proteins acquired by HIV-1 Host protein (other Strain of HIV-1 name) Producer cell (cellular origin)a (source)b CD3 (T3) H9 (T) IIIB (L) CD4 (T4) PBMCs, H9 (T) IIIB (L) CD5 (T1) PBMCs, H9 (T) IIIB (L) CD6 (T12) PBMCs, H9 (T) IIIB (L) CD8 (T8) Sup-T1 (T) IIIRF (L) CD11a (LFA-1) PBMCs, H9 (T), Sup-T1 (T), IIIB (L), IIIRF (L), CEMx174 (T), Jurkat (T), P1 (P) CEM.NKr (T), C8166 (T), U937 (M), AA2 (B) CD11b (Mac-1) H9 (T), U937 (M) IIIB (L) CD11c (gp 150,95) H9 (T), U937 (M), AA2 (B) IIIB (L) CD15 (Lewis-X) U937 (M) IIIB (L) CD18 PBMCs, H9 (T), Sup-T1 (T), IIIRF (L), IIIB (L), Jurkat (T), CEMx174 (T), P1 (P) CEM.NKr (T), C8166 (T) U937 (M), AA2 (B) CD19 AA2 (B) IIIB (L) CD25 (Tac) PBMCs, H9 (T) IIIB (L) CD30 (Ki-1) H9 (T) IIIB (L) CD43 PBMCs, CEMx174 (T), IIIRF (L), IIIB (L) (leukosialin, Jurkat (T), CEM.NKr (T), sialophorin) Sup-T1 (T), H9 (T), U937 (M), AA2 (B) CD44 (Pgp-1) CEMx174 (T), Sup-T1 (T), IIIRF (L) CEM.NKr (T) CD48 (Blast-1) PBMCs, H9 (T), U937 (M) IIIB (L) CD54 (ICAM-1) PBMCs, H9 (T), C8166 (T), IIIB (L), IIIRF (L), U937 (M), AA2 (B), LAI (L), NL4-3 (L), RAJI-CD4 (B) P1(P), 334 (P), 336 (P), 438 (P) CD55 (DAF) PBMCs, H9 (T), U937 (M), NL4-3 (L), IIIB (L), AA2 (B) JC50 (P), MG 47 (P), MP49 (P), PE51 (P), CJ48 (P) CD59 (Protectin, PBMCs, H9 (T), U937 (M), NL4-3 (L), IIIB (L), Mac inhibitor) AA2 (B) JC50 (P), MG 47 (P), MP49 (P), PE51 (P), CJ48 (P) CD63 PBMCs, H9 (T), Jurkat (T), IIIB (L), IIIRF (L) Sup-T1 (T), CEMx174 (T), CEM.NKr (T) CD71 (Transferrin PBMCs, CEMx174 (T), IIIRF (L), IIIB (L) receptor) Sup-T1 (T), H9 (T), U937 (M), AA2 (B) CDw108 (GR2) H9 (T), U937 (M), AA2 (B) IIIB (L) Cyctophilin A CEMss (T), HeLa (E), NL4-3 (L), COS (F) HXB2 (L) HXBH10 (L), IIIMN (L) Cytosketetal H9 clone 4 (T), CEMss (T) IIIMN (L) proteins PBMCs, RAJI-CD4 (B) LAI (L), IIIRF (L), HLA-DP, -DQ NL4-3 (L), 334 (P), 336 (P), 438 (P)
Abbreviations: HIV-1: human immunodeficiency virus type 1; HLA-DP and DQ, human leukocyte antigen loci DP and DQ; PBMCs, peripheral blood mononuclear cells.
aCellular origin: B, B-lymphoblastic; E, epithelial-like; F, fibroblast-like; M, monocytic; T, T lymphoblastoid. bSource: L, laboratory isolates of HIV-1; P, primary isolates of HIV-1.
This invention provides new formulations having the capacity of targeting an infectious agent, which has acquired at least one host membrane protein during its life cycle in its free form, as well as targeting the host cell itself.
This invention provides formulations which comprise ligand-conjugated vesicles that may be administered alone or in combination with a drug, for the purpose of treating or for alleviating the symptoms of a disease caused by the infectious agent. Furthermore, the ligand-conjugated vesicles could be incorporated in a gel or any other carrier to prevent or treat any disease of mucosae and/or skin. These formulations could also be used as carriers of passive vaccines against infections caused by pathogens or any disease. The drug may be incorporated into the vesicles or administered concurrently.
In a specific embodiment, the host cell is a lymphoid cell or a cell of the reticuloendothelial system. In still a more preferred version of this invention, the infectious agent is HIV. It is however clear that this invention can be applicable to any host cell infected by any infectious agent which has a life cycle such that host membrane proteins are acquired during the budding process or the construction of its external constituents. For example, a liver cell susceptible to a hepatitis virus, or a mucosal epithelial cell susceptible to a herpes virus, are versions considered within the scope of this invention.
The targeted host membrane protein is any one which is expressed by the whole cell, and acquired by the infectious agent. This protein may be selected from a histocompatibility complex protein, a membrane ATPase, thy-1, an interleukin receptor, annexin II, β2-microglobulin, CD3 (T3), CD4 (T4), CD5 (T1), CD6 (T12), CD8 (T8), CD11a (LFA-1), CD11b (Mac-1), CD11c (gp150,95), CD1 (Lewis X), CD18, CD19, CD25 (Tac), CD30 (Ki-1), CD43 (leukosialin, sialophorin), CD44 (Pgp-1), CD48 (Blast-1), CD54 (ICAM-1), CD55 (DAF), CD59 (protectin, Mac inhibitor), CD63, CD71 (transferrin receptor), CDw108 (GR2), cyclophilin A, and cytoskeletal proteins. The first contemplated protein candidates are those that are involved in the increased infectivity of HIV, namely HLA-DR, MHC-I and CD54.
In one version of this invention, the formulation comprises a ligand coupled to a lipid-comprising vesicle. In another version of this invention, the formulation comprises a ligand-conjugated vesicle containing a drug incorporated into the vesicles. The lipid-comprising vesicle includes nanoerythrosomes or liposomes.
The ligand may be selected from a surface antigen, an agonist, an antagonist, and an antibody molecule. In a preferred version of this invention, the ligand is an antibody molecule which is selected from a whole antibody and an antibody fragment capable of binding the protein of interest.
Also in a preferred version of this invention, the vesicle comprises a liposome, a sterically stabilized liposome (stealth liposome), and a combination thereof. The coupling of these liposome forms with an antibody molecule thus provides immunoliposomes.
The formulation may also comprise, besides immunoliposomes, conventional liposomes (sterically stabilized or not), comprising an effective amount of entrapped drug.
The drug contemplated in this invention is capable of affecting the cell, via its coupling with the ligand, and its binding to a cell membrane protein, for the purpose of targeting the cell of interest. The drug can also be effective directly against the free infectious agent. In so far AIDS is the disease to be treated, the drug may be selected from AZT, ddI, ddC, 3TC, indinavir, saquinavir, ritonavir, nelfinavir, ganciclovir, foscarnet, ribavirin, amphotericin B and nystatin A.
In the most specific version of this invention, the formulation is an immunoliposome coupled to an anti-HLA-DR or anti-CD4 antibody Fab′ fragment and comprising indinavir or amphotericin B as an entrapped antiviral drug.
Liposomes of the present invention are composed of any vesicle-forming lipid. For the purpose of this invention, the term “vesicle-forming lipid” is intended to cover any amphipathic lipid having hydrophobic and polar head group moieties which can form bilayer vesicles in aqueous solutions or can be incorporated into lipid bilayers. Included in this class are phospholipids such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM) where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length and have varying degrees of unsaturation. Also included in this class are glycolipids, such as cerebrosides and gangliosides. Also included in this class are cholesterol and related sterols. Also included in this class are amphipathic lipids having a derivatized hydrophilic biocompatible polymer such as polyethyleneglycol (PEG). The PEG preferably has a molecular weight ranging from about 500 to 5000 daltons. The lipid-PEG derivative is present in a proportion of about 0.75-20%, preferably about 2.5-10%. The liposomes of the present invention are most preferably composed of i) a mixture of diacylphosphatidylcholine and diacylphosphatidylglycerol (in a molar ratio ranging between 10:1 and 1:1) wherein the acyl chains are either saturated or unsaturated and have between 14 and 18 carbon atoms in length. The liposomes of the present invention also include immunoliposomes, defined herein as, liposomes which are modified by the coupling of antibody molecules which enhance the targeting of specific cells. The liposomes of the present invention also include pH-sensitive liposomes, heat-sensitive liposomes, target-sensitive liposomes and any other type of liposomes that could be used for this purpose. This invention also covers any combination of liposomes and/or drugs.
The preferred formulations of liposomes comprise dipalmitoylphosphatidylcholine (DPPC):dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 10:3 (thereafter illustrative of conventional liposomes). In another embodiment, formulations of liposomes comprise diacylphosphatidylcholine:diacylphosphatidylglycerol in a molar ratio of about 10:3. In another embodiment, formulations of liposomes comprise a mixture of diacylphosphatidylcholine:diacylphosphatidylglycerol:diacylphosphatidylethanol-amine-polyethyleneglycol in a molar ratio of 10:3:0.1-3. In another embodiment, formulations of liposomes comprise a mixture of dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 10:3 or distearoylphosphatidylcholine:distearoylphosphatidylglycerol in a molar ratio of 10:3. In another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:distearoylphosphatidylethanolamine-polyethyleneglycol (DSPE-PEG) in a molar ratio of 10:3:0.83 (thereafter illustrative of sterically stabilized liposomes or stealth liposomes). In another embodiment, formulations of liposomes comprise a mixture of dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylglycerol:dipalmitoylphosphatidylethanolamine-polyethyleneglycol in a molar ratio of 10:3:0.33 or dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylglycerol:distearoylphosphatidylethanolamine-polyethyleneglycol in a molar ratio of 10:3:0.83. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:dipalmitoylphosphatidylethanolamine-N-(4-(p-maleimidophenyl)butyryl)(DPPE-MPB) in a molar ratio of 10:3:0.33 bearing anti-HLA-DR Fab′ fragments (thereafter illustrative of anti-HLA-DR immunoliposomes). In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DPPE-MPB in a molar ratio of 10:3:0.33 bearing anti-CD4 Fab′ fragments (thereafter illustrative of anti-CD4 immunoliposomes). In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-polyethyleneglycol-MPB (DSPE-PEG-MPB) in a molar ratio of 10:3:0.83 bearing anti-HLA-DR Fab′ fragments (thereafter illustrative of sterically stabilized anti-HLA-DR immunoliposomes or stealth anti-HLA-DR immunoliposomes). In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-PEG-MPB in a molar ratio of 10:3:0.83 bearing anti-CD4 Fab′ fragments (thereafter illustrative of sterically stabilized anti-CD4 immunoliposomes or stealth anti-CD4 immunoliposomes). MPB is a coupling agent that may be replaced with any other suitable molecule for the purpose of attaching a ligand to a lipid or lipid derivative.
Other preferred formulations of liposomes comprise DPPC:DPPG in a molar ratio of 10:3 and contains amphotericin B as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG in a molar ratio of 10:3 and contains indinavir as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-PEG in a molar ratio of 10:3:0.83 and contains amphotericin B as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-PEG in a molar ratio of 10:3:0.83 and contains indinavir as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG-DPPE-MPB in a molar ratio of 10:3:0.33 bearing anti-HLA-DR Fab′ fragments and contains amphotericin B as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG-DPPE-MPB in a molar ratio of 10:3:0.33 bearing anti-HLA-DR Fab′ fragments and contains indinavir as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG-DPPE-MPB in a molar ratio of 10:3:0.33 bearing anti-CD4 Fab′ fragments and contains amphotericin B as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG-DPPE-MPB in a molar ratio of 10:3:0.33 bearing anti-CD4 Fab′ fragments and contains indinavir as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-PEG-MPB in a molar ratio of 10:3:0.83 bearing anti-HLA-DR Fab′ fragments and contains amphotericin B as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-PEG-MPB in a molar ratio of 10:3:0.83 bearing anti-HLA-DR Fab′ fragments and contains indinavir as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-PEG-MPB in a molar ratio of 10:3:0.83 bearing anti-CD4 Fab′ fragments and contains amphotericin B as an antiviral drug. In still another preferred embodiment, formulations of liposomes comprise DPPC:DPPG:DSPE-PEG-MPB in a molar ratio of 10:3:0.83 bearing anti-CD4 Fab′ fragments and contains indinavir as an antiviral drug.
The present invention is described herein below by way of specific examples and appended figures, whose purpose is to illustrate the invention rather than to limit its scope.
Any antimicrobial, bactericidal, virucidal, chemotherapeutic, antiinflammatory, antineoplastic, immunomodulator or any other agent or combination of them which is effective to treat infection and/or disease is under the scope of this invention. The term “drug” also refers to cytokines or antigens that could stimulate an immune response that would lead to an improved treatment against the said infection and/or disease.
The preparation of liposomes in the present invention can be carried out by a variety of techniques such as those described in the literature. Formulations of liposomes of the present invention include those having a mean particle diameter of any size prepared with any drug/lipid molar ratio. Incorporation of drugs into liposomes can be achieved by one or more methods of active and/or passive loading such as those described in the literature. Details for the preparation of liposomes are provided in the following examples.
Examples Involving our Liposomal Formulations for Treatment of Infection
The following examples are intended to demonstrate the preparation of liposomal formulations that could be efficient to treat infection caused by any pathogen and/or any disease, but are in no way intended to limit the scope thereof.
Preparation of Immunoliposomes
Hybridomas producing monoclonal antibodies directed against human HLA-DR (clone 2.06, IgG1), murine HLA-DR (clone Y-17, IgG2b) and human CD4 (clone OKT4, IgG2b) (American Type Culture Collection, Rockville, Md.) were isolated from ascites fluids of BALB/c mice and purified using a protein-G affinity column. Antibodies were sterilized on 0.22 μm low binding protein filters and stored at −20° C. in phosphate buffered saline (PBS, pH 7.4). Purity of antibodies was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions.
F(ab′)2 fragments of the 2.06 antibody were produced using an Immunopure IgG1 Fab′ and F(ab′)2 preparation kit (Pierce, Rockford, Ill.). In brief, the antibody was concentrated with a Centricon-100 (Amicon, Beverly, Mass.), resuspended in 0.5 ml of PBS and added to 0.5 ml of Immunopure IgG1 mild elution buffer containing 1 mM cysteine. The solution was then incubated with an immobolized ficin column for 40 h at 37° C. The solution was then eluted with 4 ml of Immunopure binding buffer and fragments were separated on an Immunopure protein A column. The column retained Fc fragments and undigested IgG1 whereas F(ab′)2 fragments were collected. Fractions containing F(ab′)2 were determined from absorbance readings at 280 nm and pooled together. The F(ab′)2 fragments (110 kD) were then concentrated using Centricon-50 and resuspended in phosphate-EDTA buffer (100 mM sodium phosphate and 5 mM EDTA, pH 6.0). F(ab′)2 fragments of Y-17 and OKT4 antibodies were produced following incubation of the antibodies with lysyl endopeptidase (in 50 mM Tris-HCl, pH 8.5) in an enzyme/substrate molar ratio of 1:50 for 3 h at 37° C. Lysyl endopeptidase cleaved IgG2b at Lys 228E/Cys 229 without perturbing disulfide bridges. The digestion products contained undigested IgG, F(ab′)2 and Fc fragments. The enzyme was removed by gel chromatography on a Sephadex G-25M column and fragments were fractionated with a protein A affinity chromatography column and resuspended in phosphate-EDTA.
F(ab′)2 fragments were incubated with 0.05 M of 2-mercaptoethylamine-HCl for 90 min at 37° C. under nitrogen atmosphere. This product cleaves the disulfide bridges between the heavy chains but preserves the disulfide linkages between the heavy and light chains. The solution was eluted on a Sephadex G-25M column pre-equilibrated with buffer and Fab′ fragments were collected. Fractions containing Fab′ were determined using a BCA protein assay reagent kit and pooled together. Fab′ fragments were concentrated using Centricon-10, resuspended in buffer and kept under nitrogen atmosphere at 4° C. until coupling to liposomes. Purity of Fab′ fragments was assessed by SDS-PAGE and their antigenic specificity was verified by flow cytometry using appropriate cells.
Liposomes of specific lipid compositions were prepared according to the method of thin lipid film hydration. In brief, the lipid mixture was dissolved in chloroform in a round-bottomed flask and the organic solvent was evaporated to form a thin lipid film. The lipid film was then hydrated with a buffer containing or not a drug to be encapsulated. In some experiments, a small proportion of radiolabelled lipids and drugs was added as radioactive tracers. Multilamellar vesicles were then sequentially extruded through polycarbonate membranes of defined pore sizes using a stainless steel extrusion device (Lipex Biomembranes, Vancouver, BC). Unencapsulated drug was removed by centrifugation of the liposomal preparation through a coarse Sephadex G-50 column and efficiency of drug entrapment was determined by radioactive countings. The mean vesicle size of the liposomes was evaluated with a submicron particle analyzer. For the coupling procedure, liposomes were incubated with freshly prepared Fab′ fragments overnight at 4° C. under continuous agitation and under nitrogen atmosphere. Uncoupled Fab′ fragments were removed through a Sepharose CL-4B size exclusion column (Sigma, St-Louis, Mo.) and the total amount of Fab′ conjugated to liposomes was evaluated using a Coomassie protein assay reagent (Pierce, Rockford, Ill.).
Even though the following examples describe specific liposomal formulations, it is deemed that a family of liposomal formulations can be easily derived therefrom, without affecting the valuable properties thereof. Formulations of liposomes of the present invention include those having a mean particle diameter of any size. The formulations of liposomes of the present invention also include those prepared with any drug/lipid molar ratio. The following examples are intended to demonstrate specific liposomal formulations of drugs which could be very efficient for the treatment of infections caused by HIV, but are in no way intended to limit the scope thereof.
In Vitro Binding and Specificity of Immunoliposomes
The binding and specificity of conventional liposomes and anti-HLA-DR immunoliposomes were evaluated in different cell lines by flow cytometry assay. In brief, cells were maintained in complete culture medium of RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin G and 100 μg/ml streptomycin at 37° C. under a 5% CO2 atmosphere. Samples were washed with PBS and resuspended. The specificity of conventional liposomes and anti-HLA-DR immunoliposomes for the cells was determined by flow cytometry from the fluorescence associated to Dil (fluorochrome incorporated into the lipid membrane).
The effect of liposomal concentration on the levels of binding of anti-HLA-DR immunoliposomes on B lymphocytes has also been investigated using two different fluorescent markers: i) a goat anti-mouse IgG (FITC) which binds to anti-HLA-DR Fab′ fragments and ii) a fluorescent lipophilic Dil marker incorporated within the lipid membrane of immunoliposomes. Results showed that the binding level of anti-HLA-DR immunoliposomes with B cells, when incubated at 37° C., rapidly saturated when using FITC conjugated goat anti-mouse IgG as a marker whereas it increased linearly over the lipid concentration range when considering the fluorescence signal associated to Dil (
Tissue Distribution Studies
The accumulation of conventional and murine anti-HLA-DR immunoliposomes within lymphoid and non-lymphoid tissues has been investigated in mice. In brief, a single bolus injection of conventional liposomes, sterically stabilized liposomes, anti-HLA-DR immunoliposomes or sterically stabilized anti-HLA-DR immunoliposomes containing a small amount of radioactive lipid was administered subcutaneously in the upper back below the neck of female C3H mice (18-20 g; Charles River Breeding Laboratories, St-Constant, QC). At specific time, animals were sacrificed and blood was collected and separated by centrifugation. At the same time, selected tissues were collected, washed in PBS and weighed. Tissues and plasma were then treated with tissue solubilizer and decoloured in H2O2. Lipid levels in all samples were monitored by counting radioactivity. Six animals were used for each time point.
Table 2 shows the area under the curve of anti-HLA-DR immunoliposomes and conventional liposomes in these different tissues. When compared to conventional liposomes, the subcutaneous administration of anti-HLA-DR immunoliposomes resulted in a 2.9 and 1.6 times greater accumulation in the cervical and brachial lymph nodes, respectively. On the other hand, the liposomal accumulation in the liver was similar for both liposomal preparations, whereas an approximately two-fold decreased accumulation was observed for anti-HLA-DR immunoliposomes in the spleen. In addition, results clearly showed that the subcutaneous administration route was very efficient for lymph node targeting as evidenced by the much higher accumulation of anti-HLA-DR immunoliposomes in these tissues when compared to that observed in the liver and spleen.
Area under the curve of anti-HLA-DR immunoliposomes and conventional
liposomes in different tissues following a single subcutaneous
administration to C3H micea.
Cervical lymph nodes
Brachial lymph nodes
aValues expressed in μmoles lipids/g tissue/h, were calculated from the mean values of the tissue distribution profile using the trapezoidal rule.
The coupling of polyethyleneglycol (PEG) on the surface of liposomes is known to increase their ability to move through the lymph after subcutaneous injection and to decrease the rate of uptake into the MPS, increasing their residence time within plasma and/or lymph. Consequently, we have evaluated if attachment of anti-HLA-DR Fab′ fragments to the end termini of PEG-coated liposomes could further improve their tissue accumulation compared to non-targeted sterically stabilized liposomes. Results showed that the concentration of both immunoliposomal formulations was higher in brachial and cervical lymph nodes than in other tissues suggesting that subcutaneous administration of immunoliposomes accumulates preferentially in regional lymph nodes (
Table 3 shows the area under the curve of sterically stabilized anti-HLA-DR immunoliposomes and of anti-HLA-DR immunoliposomes in different tissues. Results clearly demonstrated that sterically stabilized anti-HLA-DR immunoliposomes accumulate much better than conventional anti-HLA-DR immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system.
Area under the curve of sterically stabilized anti-HLA-DR
immunoliposomes and conventional anti-HLA-DR immunoliposomes in
different tissues, following the administration of a single subcutaneous
dose to C3H mice.a
Cervical lymph nodes
Brachial lymph nodes
Mesenteric lymph nodes
Inguinal lymph nodes
Popliteal lymph nodes
aValues, expressed in μmoles lipids/g tissues/h, were calculated from the mean values of the tissue distribution profile using the trapezoidal rule.
In another experiment, the concentration of the four types of liposomes (conventional liposomes, sterically stabilized liposomes, anti-HLA-DR immunoliposomes and sterically stabilized anti-HLA-DR immunoliposomes) was determined at 48 and 120 h after their subcutaneous administration to C3H mice. Results showed that the presence of PEG on the surface of conventional liposomes or anti-HLA-DR immunoliposomes had no effect on their lymphatic uptake in regional lymph nodes (brachial and cervical), but significantly increased their accumulation in other lymph nodes compared to conventional liposomes or anti-HLA-DR immunoliposomes (
We have next evaluated if the presence of anti-HLA-DR Fab′ fragments at the end termini of PEG chains affect the tissue localization of liposomes. In this set of experiments, liposomal lipids (2.5 mg/ml) were first incubated with 10 μg/ml of Dil under darkness for 1 h at 60° C. with agitation. Unbound Dil was removed by centrifugation (300×g for 15 min at 4° C.) of the liposomal preparation through a coarse Sephadex G-50 column. A single bolus injection of sterically liposomes and sterically stabilized anti-HLA-DR immunoliposomes was administered subcutaneously in the upper back below the neck of female C3H mice. At specific times post-injection (24, 48 and 120 h), animals were sacrificed and selected tissues were removed. Tissues were then washed in PBS, embedded in OCT, frozen in liquid nitrogen and stored at −20° C. Tissue sections of 10 μm thickness were cut using a Jung Figocut 2800E from Leica Canada Inc. and deposited on slides pre-treated with 2% aminoalkysilane. Coated slides were immediately observed using a fluorescence microscope and pictures were taken. Dil fluorescence was observed with a rhodamine optics excitation filter.
The tissue distribution of free indinavir and sterically stabilized anti-HLA-DR immunoliposomes containing indinavir, has also been evaluated after a single subcutaneous administration to C3H mice (
In Vivo Toxicity Studies
The toxicity of indinavir, free and incorporated in sterically stabilized liposomes, has been evaluated after 10 consecutive subcutaneous injections to mice from histopathology studies and measurements of hepatic enzymes. In brief, female C3H mice (18-20 g) (group of 10 mice) were injected subcutaneously with 500 μl of indinavir, free or incorporated in sterically stabilized liposomes (34.3 mg indinavir/kg; 540 mg of lipids/kg body weight/day), once daily for 10 days and allowed to recover for 14 days. The choice of this dose was based on daily doses administered to HIV-infected patients. Animals treated with PBS and with a 0.86% DMSO solution were used as controls. Blood samples were collected on days 0, 11 and 24 biochemical analysis. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were monitored in serum samples. On days 11 and 24, 5 mice/group were sacrificed and histopathological studies were performed on liver and spleen. In brief, tissues were fixed in a solution of 10% formalin for 4 days. After fixation, tissues were dehydrated and embedded in paraffin. Sections (5 μm thick) were placed on on gelatin-coated slides and allowed to dry for 4 h at 37° C. Slides were deparaffinized in toluene and hydrated in a series of graded ethanol solutions. During hydration, residual formalin was neutralized in ethanol-saturated picric acid. Slides were stained with hematoxylin-eosin and tissues were dehydrated, cleared and mounted with Permount (Fisher Scientific, Montréal, QC).
No significant differences in the levels of hepatic enzymes were observed at the end of the treatment (day 11) and after the two weeks recovery (day 24) when compared to baseline and control untreated mice (
TABLE 4 Area under the curve of free indinavir and sterically stabilized anti-HLA-DR immunoliposomes containing indinavir in tissues after a single subcutaneous administration C3H mice.a Ratio immuno- Immunoliposomal Free liposomal/ Tissues indinavir indinavir free indinavir Cervical lymph nodes 523.2 7.6 68.8 Brachial lymph nodes 617 4.9 126.0 Mesenteric lymph nodes 192.8 6.4 30.1 Inguinal lymph nodes 144.5 4.1 35.2 Popliteal lymph nodes 134.2 4.5 29.8 Liver 733.3 35.0 21.0 Spleen 211.3 5.3 39.9 Plasma 77.8 2.3 33.8 aValues, expressed in μmoles lipids/g tissues/h, were calculated from the mean values of the tissue distribution profile using the trapezoidal rule.
The immunogenicity of conventional liposomes, sterically stabilized liposomes bearing anti-HLA-DR Fab′ fragments or complete IgG antibody (2×10−10 mol of protein/μmol lipid) or a mixture of sterically stabilized liposomes liposomes and antibodies has been investigated in rats after four repeated subcutaneous administrations given on days 0, 7, 14 and 21. in brief, blood samples were collected at different time points (day 0, 6, 13, 20 and 27) an antibody titers in serum samples were quantified by sandwich ELISA using Nunc MaxiSorp microtiters plates. In brief, plates were coated with 5 μg/ml (100 μl/well) of the tested proteins (Fab′ fragments, whole IgG or irrelevant protein, BSA) for 2 h at 37° C. Afterwards, plates were washed three times with PBS containing 0.2% Tween-20 and incubated with blocking buffer for 30 min at 37° C. Plates were washed again and serum dilutions from immunized animals were added (100 μl/well), incubated for 1 h at 37° C. and washed again to remove unbound proteins. A biotin-conjugated goat anti-rat IgG solution (100 μl/well, diluted 1:50000 in blocking buffer) was added to each well and plates were incubated for 1 h at room temperature. Unbound antibodies were washed with PBS containing 0.2% Tween-20 and horseradish peroxydase conjugated streptavidin (100 μl/well, diluted 1:4000 in PBS containing 2% BSA and 0.05% Tween-20) was added and incubated for another 30 min at room temperature. After three washes, hydrogen peroxyde 3,3′,5,5′ tetramethylbenzidine (100 μl/well) was added to the solution and after a 30 min incubation at room temperature, the reaction was stopped by adding 50 μl of a 1 M H3PO4 solution. Absorbance was read at 450 nm on a microplate reader.
In Vitro Efficacy Studies
The ability of indinavir, free or encapsulated in sterically stabilized liposomes or sterically stabilized anti-CD4 immunoliposomes, to inhibit HIV-1 replication has been evaluated in SUPT-1 cells from measurements of reverse transcriptase activity. Briefly, 1.25×105 cells were incubated with HIV-1 (strain NL4-3; 25 ng of p24) and different concentrations of free indinavir or liposomal formulations of indinavir (10 to 100 nM) in a final volume of 500 μl. Cells were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G and 100 mg/ml streptomycin. Twice a week, 50 μl of supernatant were collected and stored at −20° C. until measurements of reverse transcriptase activity and 200 μl of cell suspension was removed for cell viability assay. Thereafter, 250 μl of fresh culture medium were added in each well. Reverse transcriptase activity was measured by incubating supernatant with 10 μl of a solution A (5 mM dithiothreitol, 50 mM KCl, 0.05% Triton X-100) and 40 μl of a solution B (5 mM MgCl2, 0.5 M EGTA, 0.04 mg of poly(rA)-oligo(dT)12-18, 3 mCi 3H-TTP (40 to 70 Ci/mmol)). After an 1 h incubation period at 37° C., samples were precipitated onto glass fiber filters by using a cell harvester system and reverse transcriprase activity was measured using a liquid scintillation counter (1205 Beta-plate; Wallac Oy, Turku, Finland).
The efficacy of different formulations of amphotericin B (AmB) to inhibit cell-free T- or macrophage-tropic HIV-1 strains that have incorporated or not host-derived HLA-DR proteins has been evaluated in HLA-DR/negative (NEG) 1G5 T-cells and HLA-DR/positive (POS) Mono Mac 1 cells. In brief, 1G5 cells were infected with HIV-1NL4-3 (10 ng of p24) while Jurkat E6.1 cells were infected with luciferase-encoding pseudotyped virions (10 ng of p24) for 2 h at 37° C. in a final volume of 200 μl of complete culture medium. Afterwards, cells were washed with PBS, resuspended in 200 μl of medium and transferred to a 96-well flat-bottomed tissue culture plate and allowed to grow for 72 h at 37° C. in a 5% CO2 atmosphere. In some experiments, HIV-1NL4-3 or 1G5 cells were pretreated with AmB (0-10 μg/ml) for 60 min at 37° C. in a final volume of 100 μl of complete culture medium prior to infection. In another set of experiments, AmB was added 2 h after the adsorption period and maintained in contact with cells for 72 h. Infection was monitored by measuring the luciferase activity. In brief, 100 μl of cell-free supernatant was withdrawn from each well and 25 μl of a cell culture lysis buffer were added before incubation at room temperature for 30 min. Thereafter, an aliquot of this cell lysate (20 μl) was mixed with 100 μl of luciferase assay buffer and activity was evaluated using a microplate luminometer.
HLA-DR/POS HIV-1NL4-3, HLA-DR/NEG HIV-1NL4-3 or HIV-1ADA (10 ng of p24) were treated with different concentrations of free or anti-HLA-DR immunoliposomes containing AmB (0-5 μg/ml) or with the corresponding amount of conventional liposomes or anti-HLA-DR immunoliposomes for 1 h at 37° C., in a final volume of 100 μl of complete culture medium. Afterwards, 1G5 cells were infected with equal amounts of pretreated HLA-DR1/POS HIV-1NL4-3, HLA-DR/NEG HIV-1NL4-3 (10 ng of p24) for 2 h at 37° C., in a final volume of 200 μl of complete culture medium. Mono Mac 1 cells were infected with HIV-1ADA (10 ng of p24) as described above. Cells were then washed with PBS, resuspended in 200 μl of complete culture medium, and transferred to a 96-well flat-bottomed tissue culture plate. After a 72 h incubation period at 37° C. in a 5% CO2 atmosphere, luciferase activity was monitored as described above.
Since pretreatment of HIV-1NL4-3 with AmB at a concentration of 5 μg/ml completely inhibited viral replication in 1G5 cells, we have next evaluated the capacity of anti-HLA-DR immunoliposomes containing this concentration of AmB to target and inhibit cell free virus. As expected, treatment of HIV-1 particles expressing host-encoded HLA-DR with anti-HLA-DR immunoliposomes containing AmB inhibited HIV-1 replication in 1G5 T-cells (
To investigate whether anti-HLA-DR immunoliposomes containing AmB inhibit specifically the infectivity of HIV-1 particles that have incorporated HLA-DR molecules, HLA-DR/NEG HIV-1NL4-3 and HLA-DR/POS HIV-1NL4-3 were incubated with different concentrations of free AmB and anti-HLA-DR immunoliposomes containing AmB. In contrast to HLA-DR/POS HIV-1NL4-3, AmB incorporated in anti-HLA-DR immunoliposomes had no effect on HLA-DR/NEG HIV-1NL4-3 replication at concentration between 0-5 μg AmB/ml (
We have next evaluated the efficacy of AmB incorporated in anti-HLA-DR immunoliposomes to inhibit viral replication of the macrophage-tropic HIV-1ADA in the monocyte/macrophage HLA-DR/POS Mono Mac 1 cell line. This cell line represents an appropriate model system to study HIV-1 infection as an immortalized cell line which parallels primary MDM properties such as necessary surface receptor/co-receptors mediating HIV-1 infection by M-tropic strains of HIV-1 and infection kinetics. Surprisingly, anti-HLA-DR immunoliposomes containing AmB were as efficient as free AmB to inhibit HLA-DR/NEG HIV-1ADA replication in a HLA-DR/POS cell line (
With a chronic disease such as HIV infection we need innovatives approaches that could improve the treatment of HIV-infected individuals. Therefore, more potent antiretroviral drugs or the use of alternative approaches that block the potential spread of virus within lymphoid tissues, their main reservoir, are urgently needed. The use of immunoliposomal drugs represents an innovative approach to inhibit virally-infected cells and cell-free particles diminishing, thereby, the viral load within lymphoid organs. Such site-specific drug targeting may allow less frequent administrations of antiviral agents and at lower doses than conventional therapy reducing therefore the marked toxicity actually seen in patients undergoing antiviral therapy with free drugs. In addition, the subcutaneous administration of liposomal drugs may lead to the generation of new modes of delivery in the future such as subcutaneous pumps. The incorporation of anti-HIV agents in anti-HLA-DR and anti-CD4 immunoliposomes could represent a novel therapeutic strategy to specifically target HIV-1 cellular reservoirs and cell-free virus with the ultimate goal of treating more efficiently patients afflicted with HIV/AIDS.
Methods of Formulation and Methods of Use
From the above detailed compositions or formulations, the skilled artisan will readily appreciate that a method of formulating the compositions is obviously contemplated and inherent to this invention. This method comprises the step of coupling the ligand to the lipid-comprising vesicle as disclosed and covered by the methodology used and practiced the specific examples. Further, the use of this formulation for treating a patient affected by an infectious disease, which comprises the step of administering the above compositions or formulations is also within the scope of the invention. The treatment may be either a passive immunization against the infectious agent or destablization of the membranes of the host cell or the infectious agent, or delivering the drug to the host cell or the infectious agent. Finally, this invention has been described hereinabove and it may be subject to variations without departing from the spirit and teachings of this invention. These variations are within the scope of this invention, as defined in the appended claims.
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|Citing Patent||Filing date||Publication date||Applicant||Title|
|US20050142114 *||Sep 17, 2004||Jun 30, 2005||Gieseler Robert K.||Targeted lipid-drug formulations for delivery of drugs to myeloid and lymphoid immune cells|
|U.S. Classification||424/178.1, 424/450, 514/50, 514/45, 514/49, 514/934|
|International Classification||A61K31/712, A61K47/24, A61K47/48, A61K31/7068, A61K9/127, A61K39/00, A61K31/43, A61K45/00, A61P31/18, A61K47/34, A61K31/7115|
|Cooperative Classification||A61K9/1271, A61K47/48823, Y10S514/934|
|European Classification||A61K47/48W6D2, A61K9/127B|
|Apr 12, 2002||AS||Assignment|
Owner name: INFECTIO RECHERCHE INC., CANADA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BERGERON, MICHEL G.;DESORMEAUX, ANDRE;TREMBLAY, MICHEL J.;REEL/FRAME:012823/0559
Effective date: 20020327
|Sep 22, 2011||FPAY||Fee payment|
Year of fee payment: 4


text:
ECU Physicians: Adult and Pediatric Health Care is a fully independent primary care med-peds practice. It functions as a group practice mixing full time faculty board certified in both internal medicine and pediatrics and the residents' continuity clinic practices. Residents have continuity clinic at the med-peds clinic starting immediately during their intern year.
Our clinic serves a great mix of patients.
Our clinic offers all aspects of primary care including preventive care, acute care, chronic disease management and typical office based procedures including joint injection, simple dermatologic procedures, circumcision, and cryotherapy. Residents receive training in all of these areas and all of these procedures.
Med-Peds Clinic Building - Moye II

domain: health


text:
Engineering GPCR signaling pathways with RASSLs.
We are creating families of designer G protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (G(s), G(i) and G(q)). We review these advances here to facilitate the use of these powerful and diverse tools.

domain: medical


text:
Recipe: Le vrai coq au vin
Total time: 2 1/2 hours, plus marinating time
Servings: 6 to 8
Note: Adapted from “The Country Cooking of France”
1 onion, sliced
1 carrot, sliced
2 celery stalks, sliced
3 garlic cloves, divided
1 teaspoon peppercorns
1 (750-ml) bottle red wine
Salt and black pepper
5 to 6 pounds chicken legs and thighs
1 tablespoon olive oil
1 (6-ounce) piece of lean smoked bacon, or thick-sliced bacon
1 tablespoon vegetable oil
3 tablespoons flour
2 cups chicken broth, more if needed
2 shallots, chopped
1 large bouquet garni of 2 bay leaves, 4 sprigs thyme and 5 to 6 stalks parsley tied together with kitchen twine
2 tablespoons butter, or more if needed
16 to 18 baby onions, peeled
1/2 pound button mushrooms, trimmed and quartered if large
1 tablespoon chopped fresh parsley
1. In a medium saucepan, bring the onion, carrot, celery, 1 clove garlic (peeled), peppercorns and wine to a boil and simmer 5 minutes, and then cool the marinade completely.
2. Rub each piece of chicken with a pinch each of salt and pepper. Pack the pieces in a deep, non-metallic bowl and pour the cooled marinade and flavorings over them. Spoon the olive oil over to keep the chicken moist. Cover with plastic wrap and let the chicken marinate in the refrigerator for at least a day, turning the pieces from time to time, and up to 3 days if you like a full-bodied flavor of wine.
3. Remove the chicken pieces from the marinade and pat them dry with paper towels. Strain and reserve the marinade liquid, keeping the vegetables separate. Heat the oven to 325 degrees. Cut the bacon into lardons -- small cubes or sticks. If you’re using thick-sliced bacon, cut the bacon crosswise into thin sticks.
4. Heat the oil in a braising pan and fry the lardons until browned and the fat runs. Transfer the lardons to a bowl using a slotted spoon and set aside for the garnish.
5. Add the chicken pieces to the pan, skin-side down, and sauté over medium heat until well browned, at least 10 minutes. Turn, cook until the other side browns, 3 to 5 minutes, and remove them. Do not overcrowd the pan; if necessary, fry the chicken in two batches.
6. Add the reserved vegetables from the marinade to the pan and fry until they start to brown, 5 to 7 minutes. Stir in the flour and cook over high heat, stirring until it browns, 2 to 3 minutes. Pour in the marinade liquid and bring to a boil, stirring constantly until the sauce thickens. Simmer 2 minutes, then stir in the broth with the shallots, the remaining two garlic cloves (chopped) and bouquet garni.
7. Return the chicken to the pan, pushing the pieces down under the sauce. Cover the pan and cook in the oven, turning the chicken occasionally, until the pieces are tender and fall easily from a two-pronged fork, about 40 minutes to 1 hour. Some pieces may be done before others -- if so, remove them so they do not dry out from additional cooking, and continue cooking the rest.
8. Meanwhile cook the garnish. Melt the butter in a frying pan, add the onions, sprinkling one-eighth teaspoon salt and a pinch of pepper, and brown them over medium heat, about 5 to 7 minutes. Shake the pan from time to time so they color evenly. Lower the heat, cover and cook the onions, shaking the pan occasionally, until just tender, 8 to 10 minutes more. Remove them with a slotted spoon and add to the reserved lardons. Add the mushrooms to the pan, sprinkling a pinch each of salt and pepper and add a little more butter if needed. Sauté until tender, 3 to 5 minutes. Add them to the lardons and onions.
9. When the chicken is cooked, remove the pieces from the sauce and set them aside. Strain the sauce into a bowl, discarding the vegetables and seasonings. Use a ladle to skim any fat on the surface. Wipe out the pan and add the garnish. Stir in the sauce; if it seems thick, add a little more broth. If it’s too thin, reduce over high heat. Taste and adjust the seasoning. Add the chicken pieces, pushing them well down into the sauce, and heat gently 3 to 5 minutes so the flavors blend. Coq au vin improves if you keep it, covered, in the refrigerator at least a day and up to 3 days so the flavor mellows before serving.
10. To serve, reheat the chicken with the garnish and sauce on top of the stove. Transfer the chicken pieces to a serving dish or individual plates and spoon over the garnish with a little sauce. Sprinkle the chicken with chopped parsley and serve any remaining sauce separately.
Each serving: 566 calories; 46 grams protein; 11 grams carbohydrates; 2 grams fiber; 30 grams fat; 9 grams saturated fat; 179 mg. cholesterol; 692 mg. sodium.
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domain: food


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text:
| Description: This piece took me some time to do as it was sort of break away from my standard color choice at the time and I was going for bright loud colors. Which was the style of a girl in my class, Keshia was always doing on her beautiful artwork. So this was homage to her work.
Though at the time I was thinking to capture black you use black, but now I know. NEVER use black. Always use combinations of two or three colors because straight black will always flatten and image. Hence why I tried fixing it by adding in white to lighten it up and break up the black but it never did work. Well you learn from your mistakes.
This piece has been featured in two art shows. One in Grand Rapids and the other at Phoenix. The Grand Rapids show was at Grand Rapids Community College in a student art show that ran three weeks. The Phoenix showing was at University of Advancing Technology for five weeks.

domain: artistic


text:
Friday, June 12, 2009
Both seniors, Myolie Wu and Skye Chan attended and encouraged the 10 participants.
Myolie demonstrated the Cat Walk, full of confidence and beauty
During the Press Conference today, the organizers invited two seniors, Myolie Wu and Skye Chan to attend and give support to the contestants, sharing about how they felt when they participated in Miss Hong Kong.
Myolie advised the participants that when they step on stage, they have to appear very confident and believed that they are the prettiest one. During photo taking, have to lower their head a little, or else they will end up like her, causing a joke, thinking that she had to confidently looked up, which resulted that she looked like she just put on 10 kilograms.
Myolie and Skye had seen the performance of the 10 participants, and even demonstrated the cat walk. Looking at Myolie's confidence and Skye's real self, it was the best demonstration for the participants.
This time, the Miss HK team will be heading to Hokkaido for filming. Myolie and Skye will be there as special guests. The participants have to take the opportunity to learn from the two seniors.
Credits: linnette_zj @ http://myolie-wu.8forum.net

domain: news


text:
My approach to psychotherapy is individually designed to enhance personal growth and mastery over problems providing freedom to enjoy life....Read More
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Likelihood of recommending Dr. Schwartz V to family and friends is 4.2 out of 5
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My experience includes an ongoing continuing education in the areas of depression,anxiety,substance abuse and problems in relationships.
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My approach to psychotherapy is individually designed to enhance personal growth and mastery over problems providing freedom to enjoy life.
The essential philosophy that guides my practice is to light one candle rather than to cry in the darkness. This includes cognitive behavioral methods to reduce pain and increase personal effectiveness.


Rock Space AC2100 Tri-Band Mesh Wi-Fi System Review
Designed for homes up to 6,000 square feet, the Rock Space AC2100 Mesh System ($ 249.99) is a tri-band Wi-Fi system based on 802.11ac (Wi-Fi 5) technology. It uses three discrete nodes that are easy to install and manage, and it comes with basic parental controls. It is enough effort, but you can find better performing systems. You’ll pay a bit more for our Editors’ Choice award winner, the Asus ZenWiFi AC CT8, but in return, you get better overall performance and more features.
No USB or multi-Gig LAN ports
The Rock Space AC2100 system uses three identical cube-shaped nodes to provide up to 6,000 square feet of coverage. The box includes the nodes, three power adapters, a LAN cable and a user guide.
The nodes are squares, measuring 3.9 x 3.9 x 3.9 inches (HWD). They are matte black, with a dark gray fabric covering on top. A small LED indicator that passes through the fabric lights up green when there is a good connection, yellow when the connection is good, and red when the connection is lost.
The base of each node contains one gigabit LAN / WAN port, two gigabit LAN ports, a reset button, and a power outlet. You don’t get USB ports with this system like you do with the Asus ZenWiFi AC CT8, nor do you get multi-gig connectivity for wired clients.
The Rock Space AC2100 is a tri-band system powered by a 900 MHz dual-core processor and 256MB DDR3 RAM. It is capable of achieving maximum (theoretical) data rates of up to 300 Mbps on the 2.4 GHz band and up to 867 Mbps on each of the 5 GHz bands. That’s a total maximum of 2,034 Mbps, hence the designation AC2100.
The system does not have Wi-Fi 6 (802.11ax) technology, instead using the older Wi-Fi 5 (802.11ac) protocol. But it does support modern intricacies such as MU-MIMO (simultaneous data broadcasting), beamforming (direct data transmissions to the client) and band steering (the router decides which bands to use for optimal performance) .
The Rock Space system is managed using the RS WiFi mobile app for iOS and Android devices. The app offers basic parental controls that allow you to suspend internet access for specific users and their devices, as well as create access schedules. However, you don’t get the age-appropriate web filters and usage reports you get with the Asus ZenWiFi AC CT8 and Gryphon AX Mesh systems. It also lacks security scanning tools that protect your network and all your connected users from malware and virus attacks.
Wireless settings are also limited. You can change your network name and password, but there are no security (encryption) or channel selection settings, and you cannot separate radio bands either.
The RS WiFi application opens to a My WiFi screen which contains a network map of all connected nodes. There is a network speedometer at the top of the screen that displays real-time upload and download speeds and the My WiFi, Settings, and Connected devices buttons at the bottom of the screen. Tap any node icon to view its connection quality, location, IP address, and MAC address.
Tap the Connected devices button to open a screen that displays all connected devices by name. Tap a device to see which node it is connected to and how long it has been connected. The My WiFi button takes you back to the main screen wherever you are in the app, while the Settings button opens a screen where you can access the wireless settings mentioned above, create a guest network, assign rules for Port forwarding and enable Parental Controls.
Enabling the Fast Roaming setting allows the system to quickly transfer clients as they roam around the house. Meanwhile, the Capacity Driven Mode setting can be used to improve performance when more than 30 clients are connected to the network at the same time. The app offers Quality of Service (QoS) settings, but there are no app-based settings. Instead, you’re limited to adjusting overall upload and download speed goals.
The installation and configuration of the Rock Space system was easy. I turned off my modem, connected a node to it using the supplied LAN cable, and powered on the modem and node. I connected my phone to the node using my phone’s Wi-Fi settings, opened the app, and hit Setup at the bottom of the welcome screen. The app identified my internet connection type (dynamic IP) and asked me to enter a Wi-Fi name and password for the new network. After about 30 seconds, the new network was created. I powered on both satellite nodes and they were automatically added to the network. To complete the installation, I gave each node a location and updated the firmware.
The Rock Space system struggled a bit to keep pace with similarly priced mesh systems in our throughput performance tests, especially when measuring the performance of the main router. The score of the 358 Mbps router node on the proximity test (same room) was 77 Mbps slower than what we saw with the Meshforce M7 router and 143 Mbps slower than the EnGenius ESR580 router. The Asus ZenWiFi AC CT8 router had a high score of 549 Mbps, which is 191 Mbps faster than the Rock Space. Likewise, the Rock Space router score of 183 Mbps at a distance of 30 feet was behind the Meshforce M7 router (205 Mbps) and the EnGenius ESR580 router (198 Mbps), while the Asus ZenWiFi AC CT8 led with a score of 228 Mbps.
The Rock Space did better on our satellite node tests, where speeds are often (but not always) slower than the main router node. It delivered 310 Mbps on the nearby drive, which was faster than the EnGenius ESR580 node (222 Mbps), but a bit slower than the Meshforce M7 node (323 Mbps). The Asus ZenWiFi AC CT8 node leads the pack, with a score of 441 Mbps. The Rock Space Knot also showed good reach in our 30ft test. Its score of 303 Mbps beats the Meshforce M7 node (298 Mbps) and the EnGenius ESR580 node (178 Mbps), but not the Asus ZenWiFi AC CT8 (329 Mbps).
In addition to measuring flow speeds, we test wireless signal strength with an Ekahau Sidekick diagnostic device and Ekahau’s Survey software. (Ekahau is owned by Ziff Davis, the parent company of PCMag). This combo generates a heat map with the signal strength represented by colors. Dark green areas on the map indicate stronger signal readings, and lighter green and yellow areas indicate weaker signal. The circles represent the location of the router and the satellite node.
As the map shows, the Rock Space system provided a relatively strong Wi-Fi signal throughout most of our test house, but the signal became weaker in the lower left bedroom.
Lacks some features
The Rock Space AC2100 tri-band mesh system offers easy installation and a pleasing aesthetic, but it has poor performance and limited functionality. The system comes with parental controls, but they are very basic and lack the granular controls you get with other systems.
You’ll spend around $ 80 more for the Asus ZenWiFi AC CT8 mesh system, but the extra cash buys you stronger parental controls, lifetime anti-malware tools, USB connectivity, and superior performance. For these reasons, it remains our Editors’ Choice for Wi-Fi 5 mesh systems.
Rock Space AC2100 Tri-Band Mesh Wi-Fi System
The bottom line
The Rock Space AC2100 is a tri-band mesh system that is easy to install and manage, but lacks functionality and performance.
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ONIPA + KONTIKI LIVE AT THE BRUDENELL – RESCHEDULED SHOW
DJ Lubi (Soul Rebels) &
***rescheduled date from 4th September 2020***
Due to the ongoing closure of live music venues UK wide due to the COVID-19 pandemic, it is unlikely they will re-open by September. Therefore this Onipa show has been rescheduled from 4th September to Friday 30th April 2021. All tickets for the original autumn 2020 date are valid for the new 2021 date.
30.04.2021 | £8.50 ADVANCE (+stbf) | 19:30 Doors
Another great “DJ Lubi Presents” African music show comes into Brudenell Social Club’s Community Room.
Afro-Futurist music machine Onipa are the brainchild of producer, composer and multi-instrumentalist Tom Excell (the music genius behind acclaimed Afro-jazz project Nubiyan Twist), and charismatic Ghanaian vocalist Kweku Sackey ( K.O.G & the Zongo Brigade). They are joined in the 4 piece live show by bass synth from Dwayne Kilvington (Steamdown Orchestra, Wonky Logic) and drums from Finn Booth (Nubiyan Twist).
Since their explosive entry in 2018, Onipa have played over 40 shows in 10 different countries, including Into the Great Wide Open (NL), Glastonbury Festival (UK), Eurosonic (NL), Nuits Sonores (BE), Tribu Festival (FR), The Great Escape (UK), Fusion Festival (DE) and Africa Express (UK), winning support from The Guardian, Clash Magazine, Radio Nova and many more media outlets.
It’s a unique fusion of African music and electronics, drawing on a wide range of influences – Ghanaian Hi Life, Congolese Soukous, Mali Blues, South African Shangaan Electro & Township Music, Nigerian afro-disco, Moroccan Gnawa – combined with ragga, dancehall, UK dance music, jazz and hip hop and inspired by artists like Oumou Sangare, Konono No.1, Kokoko, Flying Lotus, Ebo Taylor, William Onyeabor, Kiki Gyan.
After an EP and 2 singles on Wormfood/Mawimbi, Onipa released their first full studio album “We No Be Machine” on Strut Records of London in March 2020 to great critical acclaim.
Support on the night comes from Leeds/Bradford highlife jazz combo Kontiki who have built a strong local following with their Highlife meets Afro Jazz fusion – a blend of sweet sounding West African guitars, a driving rhythm section topped off with firing sax solos.


text:
By Maurice Beaulieu, Catholic News Service
ORLANDO, Fla. — Kristen Hanson, community relations advocate for the Patient Rights Action Fund, told the emotional story of her husband’s battle with brain cancer and how the couple fought it knowing “every single day is a gift.”
She spoke during the annual Florida Culture of Life Conference, hosted by the Diocese of Palm Beach, Florida. The daylong conference Oct. 24 was a virtual gathering due to the ongoing COVID-19 pandemic.
Hers was a heartwarming and passionate presentation showing the value of preserving life and why assisted suicide in America symbolizes a nation of potential hopelessness.
Currently, seven states and the District of Columbia allow assisted suicide — a physician-aided agreement to end a patient’s life based solely on the patient qualifying for death under state law — determined to be terminally ill or otherwise incapacitated in the state’s view.
Although Hanson’s husband, James Joseph Jr., known as JJ, succumbed to cancer in 2017, his voice was heard during the presentation — “Warriors of Hope: Fighting Against Assisted Suicide in America.”
Hanson first showed two short videos of her family that were filmed before JJ died. The couple discussed their ordeal and what kept them from losing hope.
“We were living, what people would consider is, the American dream,” JJ had said in the video of the time spent with his wife and then-infant son, James. “May of 2014, I started to feel, what felt like, a deep anxiety attack and I knew something was wrong with me because it kept getting worse. At that point, my ability to speak began to go away.”
After a visit to the doctors, the Hansons discovered JJ, 33, was suffering from an aggressive form of brain cancer — glioblastoma multiform, or GBM — on his left temporal lobe.
Because of the particular placement of the cancerous tumors, doctors were hesitant to operate for fear of doing damage to JJ’s brain, such as losing the ability to speak and comprehend language.
Physicians told JJ he had roughly four months to live. In his best-case scenario, maybe a year.
“In that moment, time stood still,” Hanson said of her panic. “I couldn’t breathe. Everything went silent except for the pounding of my heart and the sound of my own voice in my head crying out why? Why only four months? … We have a baby. Our poor baby. Poor James.”
But the couple urged one another not to give up hope. JJ surpassed that four-month mark by more than three years before his death — he even went into remission for a couple of years during that time.
As the two family videos were broadcast, conference attendees could see a thumbnail photo of Hanson in the upper-right hand corner. She was watching the video just as conference visitors were. The first family video was about six minutes in length. The end of the video shows JJ and Kristen sitting together looking at one another. Kristen is heard saying, “I’m more in love with you now than I ever thought possible.”
“Every single day is a gift, and you can’t let that go,” JJ said.
The next video was shot when her husband only had weeks to live. His voice sounded different, his body was affected by his illness. But the couple continued to state how they had to “hold on to hope.”
“You cannot think about assisted suicide in one situation. You have to think about who it could hurt. … The joys we have experienced in these last three and a half years are too many to count, and when if we had given up hope we would have missed out on so much,” Hanson said before echoing her husband’s words. “Every moment we share together is a gift.”
Shortly after his diagnosis, JJ underwent surgery. “The neurosurgeon removed the two tumors and actually found a third smaller tumor,” Hanson said.
JJ’s recovery started off well. But later that week, he had difficulty speaking and answering questions. Suspecting JJ was having sub-clinical seizures, Hanson convinced the doctors to perform an EEG. She was correct. JJ had a total of 10 seizures since his operation.
“I had to hold onto hope,” she said. Bouts of radiation and chemotherapy followed. “We enrolled him in a clinical trial and thankfully, JJ went into remission for almost two years.”
In late October 2016, JJ’s cancer reemerged. Immediately after he began his new treatment, the Hansons discovered they were going to have another baby. “In July of 2017, we welcomed our little miracle baby into the world.”
That joy was short-lived when JJ’s new therapy wasn’t working as expected. Yet, they chose to enjoy life and whatever time they had together than turn toward death.
“JJ chose to fight against the ableism that permeates our society and our medical system, and which has perhaps its most deadly expression in assisted suicide policy,” Hanson said.
Hanson recalled when she and her husband saw news stories about Brittany Maynard, 29, who gained notoriety for her decision to end her own life. First diagnosed in early January 2014 and again in April with additional forms of brain cancer, Maynard was given six months to live.
A California native, Maynard moved to Oregon, where assisted suicide is legal.
“I remember when we first saw (Maynard) on TV, we could see so clearly how her story was being glamorized to perpetuate a message of hopelessness,” Hanson told the pro-life conference. “JJ looked at me with tears in his eyes and said, ‘Kris, if I had suicide pills with me in my nightstand a few weeks ago, I might have taken them. And you can’t undo that. This is so dangerous for patients like me.'”
JJ took initiative to publicize his story, she said. “JJ connected with the Patient Rights Action Fund and a few week later, PRAF came to capture our story,” Hanson said. Eventually, JJ was asked to helm the organization as its president, “leading the national fight against the legalization of assisted suicide.”
He knew the campaign that promotes assisted suicide is strong because it appeals to people who are in their most susceptible state, Hanson said. He made his wife promise that after his death, she would continue sharing the family’s story to protect the vulnerable against the threat of legalized assisted suicide.”
Hanson cited several examples why assisted suicide continues to be problematic for people told they have a terminal illness, including:
— “Doctors make mistakes” in their diagnoses.
— “Insurance companies deny care and offer a cheap death instead.”
— “Mental health conditions often go ignored and patients struggling with depression receive the medication.”
“Pain is not the reason patients end their lives by assisted suicide,” she continued. “When a person chooses assisted suicide, it is typically out of “existential suffering or disability related concerns at the end of life, such as fear of being a burden to friends and family.”
The legalization of assisted suicide sets up a discriminatory system that determines who may live and who may die, she said. “This insidious form of ableism results in death to the devalued group and nothing could be more discriminatory.”

domain: sociology


text:
Photo: Ablestock. Titusville is a town on the east coast of central Florida that attracts tourism primarily through its proximity to the NASA space complex and the nearby Canaveral National Seashore. One and a half million people visit the Seashore every year, so it is vital each one respect the environment. To protect the fragile dune structures, use only the boardwalks to pass from the parking areas to the beach. The beas are on a barrier island separated by the Intracoastal Waterway - here Mosquito Lagoon on the Indian River - from the mainland. The Canaveral National Seashore is 12 miles east of Titusville. The south beas are closed to the public three days prior to the launch of a space shuttle, and all day on launch days.
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Nicknamed the 'Gateway To Nature And Space', Titusville is perfectly located right by a lot of super spaces and landmarks, including the Indian River, the Atlantic Ocean, multiple beas, and lots of amazing natural areas like St. Johns National Wildlife Refuge and the Charles H. Bronson State Forest. Titusville lies near the longest continuous length of undeveloped beach on Florida's east coast covering 57, acres. Top 10 Titusville Sehenswurdigkeiten: Hier finden Sie Bewertungen und Fotos von Reisenden uber 33 Sehenswurdigkeiten, Touren und Ausfluge - alle Titusville Aktivitaten auf einen Blick.
With unique architecture and gorgeous seasonal decorations, these tours should be on your list of things to do this holiday season.
Read on Additional Info To read the latest magazine, visit Titusville.
Titusville Water System Maintenance to Begin October 12 Titusville water customers may notice a change in their drinking water beginning on October 12 as the city begins a chlorine dosing of its water distribution system. Florida Department of Health Encourages Residents to Get Flu Shot Now County health departments in the Central Florida region are urging residents to get vaccinated against the flu as soon as possible.
Titusville Police Keep School Children Safe We are continuing our dedication to the safety for schoolchildren by enforcing a yearlong proactive enforcement detail in school zones. Tue Oct.
If you are gay and you want to practise cruising and to have casual NSA encounters in public places in Titusville in an anonymous way, here you can find spots such as beas, parks, forests and other spaces next to urban areas, as well as every kind of public toilets and rest areas of highways where you can practise cruising in Titusville, Florida. Titusville Police Department's Officers received 50 Individual First Aid Kits (IFAK), thanks to the generosity and proactive efforts of Parrish Medical Center, Team Health Emergency Physicians and the Jess Parrish Medical Foundation. Additional Info October Titusville Talking Points Out Now! The October Issue of Titusville Talking Points is out now! To read the latest magazine. Titusville's best FREE dating site! Free Online Dating for Titusville Singles at hondaatclub.com Our free personal ads are full of single women and men in Titusville looking for serious relationships, a little online flirtation, or new friends to go out with. Start meeting singles in Titusville today with our free online personals and free Titusville chat!
Regular City Council Meeting at P. Board of Adjustments and Appeals. Lots of useful amenities can be found here including all the typical campground facilities like showers, restrooms, power hook-ups, and picnic areas.
There's a good pier for fishing and lifeguards are on-duty too, so families with young kids can feel extra safe. For an all-round Florida holiday, Titusville is an excellent place to base yourself.
Nicknamed the 'Gateway To Nature And Space', Titusville is perfectly located right by a lot of super spaces and landmarks, including the Indian River, the Atlantic Ocean, multiple beas, and lots of amazing natural areas like St.
Bronson State Forest. The Kennedy Space Center is one of the most incredible places in America, while the local beas and green spaces around the city allow us to keep our feet on the ground and enjoy the natural wonders of our own planet.
Florida is famous for many things, but the Sunshine State is best known for playing home to the Walt Disney World Resort and all four of its unique theme parks. Disney World is the most visited resort on the planet, with more than 50 million tourists jetting in from all over the globe each and every year to enjoy the thrills and spills of the various rides and attractions, as well as having a chance to meet Mickey Mouse himself and other popular Disney characters.
Of all the four theme parks, Epcot is often regarded as one of the most special. Opened inEpcot is dedicated to the theme of technological achievement and international cultures.
Like a never ending world expo, Epcot is immediately recognizable for its huge sphere standing right near the entrance and visible from almost any area in the entire park. The park is divided into two sections: Future World and the World Showcase.
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The former is focused on various achievements of mankind like space travel and technology, while the latter is divided into 11 pavilions, each representing a country from around the world. With so many rides and attractions to enjoy all around the park, Epcot offers a treasure trove and adventures and experiences for people of all ages.
It's impossible to talk about Epcot without mentioning Spaceship Earth. The oldest ride in the park, Spaceship Earth is actually located inside the iconic Epcot sphere. New visitors to the park flock here as their first port of call and it's a thrilling experience to simply step up into this impressive structure.
The ride itself takes place entirely indoors and lasts for around 15 minutes; it's a slow-moving journey through the history of human communication, beginning in the prehistoric era and flowing through to the modern day, all narrated by Oscar-winning star Dame Judi Dench. Opened inTest Track is one of only a couple of Epcot rides that were truly designed with thrill seekers in mind. The cars designed by each passenger also undergo the various tests digitally, with the results and top scores displayed for all to see.
If you've ever wanted to know how it feels to be an astronaut and jet off into space, this is the ride for you. This interactive, exciting ride opened back in and underwent a facelift in The ride uses centrifugal forces to simulate space flights, one of which goes to Mars and the other orbits the Earth.

domain: geography


text:
Detox is not the expulsion of ‘toxins’ from your system, but a reaction of your nervous system to the absence of alcohol. Withdrawal from alcohol and its sedative effects results in an overreaction of the nervous system. A number of severe symptoms can develop, such as disorientation, extreme anxiety, diarrhoea, very high blood pressure, delusions, heart rhythm changes etc. This is very dangerous if not supervised and monitored by personnel experienced in addiction recovery treatment. These symptoms can now be controlled in a safe way through proper medical attention and pharmacotherapy during the detox period. Our clinical staff has extensive experience dealing with alcohol detox and conducts the process in as much safety and as comforting an environment as possible. This is a vast improvement from a time before modern medicine when there was a death rate of 33% for those going through severe alcohol withdrawal.
Aftercare services are primarily designed to help recovering alcoholics maintain their sobriety once their treatment has finished. According to the National Center for Biotechnology Information (NCBI), a good after-care service can make a who difference to whether a person's alcoholism treatment is successful. Some states provide sober houses, which provide somewhere to live for people who are trying to stay sober. While it is not formal treatment, staff members who supervise the houses will remind individuals if they have a therapy appointment or support group meeting. They also, along with other residents, offer support and the chance to live in a completely alcohol-free environment.
According to the National Institute on Drug Abuse, addiction is a "chronic, relapsing brain disease that is characterized by compulsive drug seeking and use, despite harmful consequences."7 There is no simple cure for addiction; however, effective treatment can help you become and stay sober.7 You will have to manage your addiction throughout your life, the same way a diabetic has to manage their condition with ongoing efforts like a proper diet and exercise.8 Drug Rehab Near Me
Many patients get caught up in trying to define their relationship with drugs and alcohol. For example, drug abuse, has a far less threatening reputation than that of drug addiction. According to Medline Plus, an issue with drug abuse is defined as the regular abuse of any illicit substance including alcohol over the course of a year with negative consequences. These negative consequences can be financial, interpersonal, work-related, legal, health-related – anything that changes the patient’s experience of day-to-day life for the worse.2 How to stop a drug addiction FOREVER: #1 Real cause of addiction revealed
Nalmefene, an opiate antagonist that is similar in its chemical structure to naltrexone, is one of the most recent drugs being investigated for the treatment of alcoholism. Like naltrexone (sold as ReVia, Depade, or Vivitrol), nalmefene deprives the person struggling with substance use of the pleasurable feelings associated with drinking. But nalmefene is less toxic to the liver than naltrexone. As of 2013, nalmefene was still undergoing clinical trials through the U.S. National Institutes of Health before receiving FDA approval. From Rehab to a Body Bag | Dying for Treatment: VICE Reports (Full Length)

domain: psychology