text:
|Publication number||US5149529 A|
|Application number||US 07/773,073|
|Publication date||Sep 22, 1992|
|Filing date||Oct 11, 1991|
|Priority date||Apr 8, 1988|
|Publication number||07773073, 773073, US 5149529 A, US 5149529A, US-A-5149529, US5149529 A, US5149529A|
|Inventors||Rodney Ho, Thomas Merigan, Rae L. Burke, Dino Dina|
|Original Assignee||Board Of Trustees Of Leland Chiron Corporation, Stanford Junior University|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (16), Non-Patent Citations (12), Referenced by (38), Classifications (23), Legal Events (4)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This is a continuation of copending application Ser. No. 07/179,095, filed on Apr. 8, 1988, now abandoned.
This invention relates to recombinant DNA technology, to the treatment and prevention of disease, and to compositions therefor. In particular, this invention relates to liposomal formulations of recombinant Herpes virus surface glycoprotein D-1 (HSV rgD-1).
The herpes viruses include the herpes simplex viruses, comprising two closely related variants designated types 1 (HSV-1) and 2 (HSV-2). These types cross react strongly, but can be distinguished by neutralization titrations. HSV-1 and HSV-2 are responsible for a variety of human diseases, such as skin infection, genital herpes, viral encephalitis, and the like.
The herpes simplex virus is a double stranded DNA virus having a genome of about 150 to 160 Kb, packaged within an icosahedral nucleocapsid, enveloped in a membrane. The membrane includes a number of virus-specific glycoproteins, the most abundant of which are gB, gC, gD, and gE. The proteins gB and gD are cross-reactive between HSV-1 and HSV-2.
It is a matter of great medical and scientific interest to provide safe and effective vaccines for humans against both HSV-1 and HSV-2, and where infection has occurred, to provide therapies for treatment of the disease. One promising approach has been the use of isolated glycoproteins, which have been shown to provide protection when injected into mice subsequently challenged with live virus. These proteins are produced by, e.g., the methods taught in U.S. Pat. No. 4,618,578, incorporated herein by reference.
Another critical aspect affecting vaccine efficacy is the vehicle employed to deliver the antigen. Proper activation of the immune system depends on a complex series of interactions between the immune system's various components, which include antigen presenting cells (APC), e.g., macrophages, dermal Langerhans cells, and dendritic cells; helper thymocytes (Th -cells); cytotoxic thymocytes (Tc -cells); and immunoglobulin-secreting B-cells. APCs ingest foreign particles in the body, partially digest the antigens thereon, and "present" the partially-digested antigens on their surfaces in association with proteins encoded in the major histocompatability complex (MHC) genes. MHC proteins are classified as type I (which are expressed on the surfaces of all nucleated cells), and type II, which are expressed only on certain cells of the immune system. Type II proteins are interchangeably denoted as MHC Ia (for "immune-associated"). APCs express both MHC I and Ia, but it is the Ia-antigen complexes that are important for initial activition of the immune system. These antigen-MHC Ia complexes are recognized by Th cells that bear a T-cell receptor capable of binding to the complex. This binding, in addition to the secretion of certain cytokines (particularly interleukin-1, "IL-1"), effects activation of the Th cell. As a result, the stimulated Th cells proliferate and begin secreting other cytokines, eventually activating Tc cells and B-cells specific for the antigen that originally started the cascade. Tc cells recognize antigen in combination with MHC I proteins on cell surfaces, and effect lysis of such cells in a process known as cell mediated immunity (CMI) Thus, Tc cells and CMI are believed to be particularly important in the immune response to viral infection, as lysis of cells exhibiting viral antigens results in interruption of viral reproduction.
B-cells express antibodies of the various classes in a process known as humoral immunity. The antibody classes most important in resistance to infection are IgM, IgG, and IgA. Antibodies may also play a role in protection from viral infection, by fixing complement on infected cells, by clearing viral particles from the bloodstream, by opsonizing cells for clearance by macrophages and neutrophils, and by initiating antibody-dependent cell-mediated immunity (ADCC), which is effected by killer cells (K).
The efficacy of these immune defenses varies depending upon the pathogen's antigenicity and mode of infection. When designing a vaccine, the object is to provide for activation of those immune defenses which will result in the most effective protection from the pathogen in question. In general, an adjuvant is often employed in order to increase the immune response to a particular antigen. Most adjuvants, however, are only able to enhance humoral immunity, and fail to increase cell mediated immunity.
The mode of administration is also of critical importance. Most vaccines are administered intramuscularly or subcutaneously (in rare cases intradermally), as lymph in these tissues drains into the lymph nodes where the antigens will be accessible to Th cells and macrophages. In contrast, administration of soluble antigens by intravenous routes may result in tolerance to the antigen, i.e., a suppression of any immune response.
The archetypal adjuvant is Freund's adjuvant, which is commonly used in laboratory immunology. Freund's complete adjuvant (FCA) consists of an oil-in-water emulsion of mineral oil, surfactant, and killed mycobacteria (M. tuberculosis). Freund's incomplete adjuvant (FIA) omits the mycobacteria. These adjuvants provide an enhanced immune response, but are not suitable for administration to humans, as mycobacteria are the causative agent of tuberculosis, and may cause adverse responses detrimental to the subject. Further, the mineral oil component is not metabolized, and may cause abcesses and granulomas at the site of injection. Researchers have since replaced the mycobacteria with muramyl dipeptide (MDP) derivatives, which were determined to be the smallest component of the mycobacterial cell walls that retained the adjuvant activity. The preparation of various MDP derivatives and analogs is described in detail in U.S. Pat. Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890. It is currently believed that the MDP derivative stimulates the release of IL-1. The mineral oil component has been replaced by various synthetic and metabolizable vegetable oils. See for example Allison et al, U.S. Pat. No. 4,606,918, disclosing an adjuvant formulation comprising muramyl dipeptide, a polyoxypropylene-polyoxyethylene block polymer (Pluronic®), a non-ionic surfactant, and a metabolizable oil such as squalane. Other adjuvants known in the art include alum, lipid A, trehalose dimycolate, and dimethyldioctadecylammonium bromide (DDA). However, only alum has been approved for human administration.
Liposomes have also been employed to deliver biologically active material. See for example Allison, U.S. Pat. No. 4,053,585, which discloses the administration of several antigens in negatively-charged liposomes, optionally including killed M. tuberculosis. Fullerton et al, U.S. Pat. No. 4,261,975, discloses the use of separated influenza membranes, with hemagglutinin spikes attached, which is bound to liposomes for use in influenza vaccines. Tarcsay et al, U.S. Pat. No. 4,406,890, discloses the use of liposomes to encapsulate MTP-PE (a lipophilic MDP derivative) and bovine serum albumin (a standard model antigen). Liposomes are small vesicles composed of amphipathic lipids arranged in bilayers. Liposomes are usually classified as small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), or multi-lamellar vesicles (MLV). SUVs and LUVs have only one bilayer, whereas MLVs contain many concentric bilayers. Liposomes may be used to encapsulate various materials, by trapping hydrophilic compounds in the interior or between bilayers, or by trapping hydrophobic compounds in the bilayer.
Liposomes exhibit a wide variety of characteristics, depending upon their size, composition, and charge. For example, liposomes having a small percentage of unsaturated lipids tend to be slightly more permeable, while liposomes incorporating cholesterol tend to be more rigid and less permeable. Liposomes may be positive, negative, or neutral in charge, depending on the hydrophilic group. For example, choline-based lipids impart a positive charge, phosphate and sulfate based lipids contribute a negative charge, and glycerol-based lipids tend to be neutral in solution.
Several HSV vaccines have been prepared. S. Dundarov et al, Dev Biol Standard, 52:351-57 (1982) disclosed the treatment of humans with formalin-inactivated HSV in distilled water. GRB Skinner et al, Dev Biol Standard, 52:333-44 (1982) disclosed the treatment of humans with formalin-inactivated HSV in saline. L. Chan, Immunol, 49:343-52 (1983) disclosed the protective immunization of mice against HSV challenge by vaccination with gD in saline. Kino et al, U.S. Pat. No. 4,661,349 disclosed vaccines comprising purified HSV gB with alum. Person, U.S. Pat. No. 4,642,333 disclosed HSV gB and its administration to rabbits in Freund's adjuvant. Recently, L.R. Stanberry et al, J Infect Dis, 157:156-163 (1988) reported the use of rgD and rgB in a vaccine to ameliorate the symptoms of genital herpes infection in guinea pigs.
The instant invention comprises a method for treating infection in an individual infected with HSV-1 and/or HSV-2, by parenterally administering a composition comprising an HSV surface glycoprotein and an MDP equivalent, encapsulated in liposomes in aqueous suspension. Presently preferred glycoproteins are gB1, gD1, gB2, gD2, and derivatives thereof. Most preferred is rgD-1.
Another aspect of the invention is the composition suitable for effecting treatment of HSV infection, which comprises an effective amount of an HSV surface glycoprotein (preferably rgD-1), an effective amount of an MDP equivalent, encapsulated in liposomes in aqueous suspension.
Another aspect of the invention is a lyophilized composition comprising an HSV surface glycoprotein (preferably rgD-1), an MDP equivalent, and liposome-forming lipids, which is capable of forming liposomes upon hydration.
FIG. 1 depicts a plot of HSV-2 lesion severity vs. time for four experimental groups of animals.
FIG. 2 depicts a plot of cumulative HSV episode recurrence for four experimental groups of animals.
The term "HSV glycoprotein" refers to any of the glycoproteins found in the membrane region of HSV-1, HSV-2, and related herpes viruses. Presently preferred HSV glycoproteins are gB, gC, gD, and gE. Included within this definition are glycoproteins extracted from natural viruses (e.g., from infected sera or cell culture), and glycoproteins produced by recombinant methods. Such glycoproteins may additionally be modified, either by chemical or enzymatic means (e.g., by proteolytic cleavage), or by recombinant DNA techniques (e.g., by fusing HSV glycoprotein genes with other genes to provide fusion proteins, or by deleting or replacing sections of the DNA sequence).
The term "rgD-1" refers to a recombinantly-produced HSV surface glycoprotein D1, which is truncated at the carboxy terminus and lacks the hydrophobic membrane anchor and cytoplasmic domains of the native protein. After removal of the signal peptide in CHO cells, the resulting protein is 290 amino acids in length.
The term "MDP equivalent" refers to any of the commonly known compounds chemically related to N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), and capable of eliciting IL-1 expression in vivo. Appropriate MDP equivalents include, without limitation, MDP itself, N-acetylmuramyl-L-threonyl-D-isoglutamine ("Thr-MDP"), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine ("Nor-MDP"), n-butyl N-acetylmuramyl-L-alanyl-D-isoglutaminate (murabutide), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl-2-[1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy)]-ethylamide (MTP-PE), and the like. Other MDP eqivalents, for the purposes of this invention, include gamma interferon, IL-1, granulocyte-monocyte colony stimulating factor (GM-CSF), and monocyte colony stimulating factor (M-CSF).
The term "treatment" as used herein refers any of (i) the prevention of infection or reinfection (prophylaxis), (ii) the reduction or elimination of symptoms, and (iii) the substantial elimination of the virus.
The term "effective amount" refers to the amount of rgD-1 sufficient to induce an immune response in a recipient subject when administered in the composition of the invention. The immune response is preferably protective immunity, whether this is achieved by humoral immunity, cell mediated cytotoxicity, or other mechanisms. "Effective amount" also refers to the amount of MDP equivalent necessary to potentiate the subject's immune response to the antigen.
HSV rgD antigens may be prepared by the method set forth in U.S. Pat. No. 4,618,578, incorporated herein by reference. Briefly, the HSV gene encoding gD is cloned in a shuttle vector suitable for modification and amplification in a prokaryotic host (e.g., E. coli) and for expression in a eukaryotic vector. Standard microbiological techniques are employed to provide suitable promoter and termination regions for the gD gene, and selectable markers for the vector, in order to allow control of gD expression in the expression host. The gD gene may be supplemented with nucleotides encoding other regulatory and functional sequences, for example for secretory signal peptides, or for leader cleavage by endogenous enzymes. One may also delete segments of the gD gene: for example, one may delete the hydrophobic membrane anchor domain to improve the solubility of the protein. The rgD-1 protein preferred herein is truncated at the carboxy terminus, and is lacking both the membrane anchor and the cytoplasmic domain.
Preferably, the rgD-1 protein is expressed in Chinese hamster ovary (CHO) cells. The protein is extracted and purified from the cell culture medium by ammonium sulfate precipitation, immunoaffinity chromatography, and ultrafiltration.
MDP equivalents are prepared by methods known to those of ordinary skill in the art. For example, MDP, Nor-MDP, and Thr-MDP may be prepared following the procedures set forth in Jones et al, U.S. Pat. Nos. 4,082,735 and 4,082,736, incorporated herein by reference. Murabutide may be prepared following the procedures set forth in Derrien et al, U.S. Pat. No. 4,185,089, incorporated herein by reference. MTP-PE is prepared following the procedures set forth in Tarscay et al, U.S. Pat. No. 4,406,890, incorporated herein by reference.
Liposomes may be prepared by a large variety of methods. The simplest method comprises evaporating the solvent from a chloroform solution of lipids, leaving a lipid film on the inner surface of the vessel. An aqueous solution (usually lacking Mg++ and Ca++ ions) is then added and agitated (e.g., by vortexing) to form liposomes. Other methods involve adding lipids to aqueous solution with sonication, injection of lipid solutions into aqueous solution with evaporation of co-solvents, or use of special equipment.
In the instant case, we have achieved high capture efficiency by the following process: a mixture of lipids, preferably egg phosphatidylcholine (EPC) and phosphatidylglycerol (PG) in a 9:1 ratio, and MTP-PE (in an amount equal to the PG) is dried under a stream of N2, and vacuum desiccated for 30 minutes. The lipids are then hydrated with sterile water and phosphate-buffered saline (PBS), vortexed, then sonicated for 15 minutes to provide a translucent liposomal suspension. The suspension is then sterile filtered, the antigen added, and the mixture frozen and lyophilized overnight. For use, the lyophilized material is rehydrated, vortexed, and allowed to stand, providing an injectible suspension of SUV liposomes. Capture efficiency is estimated at 47%.
The composition is administered parenterally, usually by intramuscular or subcutaneous injection. One may also administer the composition by intravenous routes, if desired. The precise amount of composition administered will of course depend upon the size, age, and health of the subject, the extent and severity of the disease, and the exact nature of the components used in preparing the composition, hence it is not possible to provide an exact dosage. However, an effective amount of rgD-1 for a 70 Kg adult human ranges from about 10 ug/dose to about 100 ug/dose, preferably about 25 ug-50 ug/dose. An effective amount of MTP-PE ranges from about 1 ug/dose to about 100 ug/dose, preferably about 20 ug/dose. The amount of lipids used is not particularly critical, but a convenient range is about 0.05 mg-3.0 mg/dose. The precise dosage may determined by one of ordinary skill in the art. The composition may be administered in stages, as is typical for vaccines; i.e., an initial administration is followed two to six weeks later by additional "booster" administrations, in order to effect a lasting immunity.
The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.
The preferred antigen of the invention, a 290 amino acid truncated gD protein, is prepared as described below.
A library of EcoRI restriction fragments of HSV-1, strain Patton, were cloned into the EcoRI site of the plasmids pBR322 or pACY184 (obtained from Dr. Richard Hyman, Hershey Medical Center, Hershey, Pa.). The gD1 gene, which is entirely contained within a 2.9 Kb SacI fragment within the EcoRI fragment of clone H from this library, was obtained by restriction digestion, purified by gel electrophoresis, and digested to completion with HindIII and NcoI. The 5' end of the gD gene, consisting of 74 bp of 5' untranslated sequence plus 60 bp encoding the amino terminal 20 amino acids, was isolated as a 134 bp HindIII/NcoI fragment. A portion of the 3' end of the gD gene was obtained by digestion of pYHS119 (described in U.S. Pat. No. 4,618,578, incorporated herein by reference) with NcoI and SalI and isolation of the 873 bp fragment. These two fragments (5' and 3' ends) were ligated together with the plasmid pUC12 (previously digested with HindIII and SalI). The resulting plasmid was digested with HindIII, the sticky ends filled in with Klenow fragment (DNA polymerase I), and digested with SalI. The 1007 bp fragment containing the gD gene was isolated and ligated into the mammalian expression vector pSV7d, previously cut with SmaI and SalI (see R.L. Burke et al, J Biol Chem, 261:12574 (1986 )). The resulting plasmid (designated pHS132) encodes 315 amino acids out of a total of 394 for the complete gD1 protein (including the 25 amino acid signal sequence). The resulting protein is truncated at the carboxy terminus, and lacks the hydrophobic membrane anchor domain and the cytoplasmic domain. After cleavage of the signal sequences, a 290 amino acid protein (rgD-1) is secreted into the culture medium.
The rgD-1 expression vector pHS132 is cotransfected into CHO cells with the Ad-dhfr plasmid which encodes the selectable and amplifiable marker gene dihydrofolate reductase (DHFR). DHFR-positive clones were screened for synthesis and secretion of rgD-1 with an ELISA, and the most productive cell line propagated in roller bottles.
The rgD-1 truncated protein was isolated from the conditioned medium as follows:
Culture medium (300 mL) was acidified to pH 4.5 by adding 15 mL 1M citrate (pH 3.0), and allowed to stand for 10 minutes at 4° C. The acidic medium was brought to 1M (NH4)2 SO4 by adding 462 g of solid (NH4)2 SO4. The precipitate was removed by centrifugation at 5,000 rpm at 4° C. for 10 minutes. Next, 300 mL of supernatant was applied to a 15 mL column of fast flow Phenyl Phenyl "SEPHAROSE" ("SEPHAROSE"--O--CH2 --CHOH--CH2 --O--C6 H5, Sigma Chemical Co.) at pH 4.6, and eluted with a 30 minute linear gradient from 1M (NH4)2 SO4, 0.5M sodium acetate, pH 4.6 to 0.05M sodium acetate, pH 4.6. Peak fractions were identified by ELISA and were pooled. The pooled fractions were dialyzed into 0.02M sodium acetate, pH 6, and loaded onto a 5 mL "BIOGEL" TSK SP 5 PW cation exchange column (Biorad). Elution was achieved with a 60 minute gradient from 0.02 sodium acetate, pH 6.0 to 0.5M NaCl, 0.02 sodium acetate, pH 6.0. Peak fractions were identified by ELISA, analyzed by SDS PAGE, and pooled based on gel purity. Pooled fractions were then concentrated to 0.5 mL by pressure filtration and applied to a 25 mL SUPEROSE 12 GPC column equilibrated with 0.1M (NH4)2 CO3, pH 7.0. Peak fractions were identified by ELISA and by SDS-PAGE, and were pooled and stored at -20° C.
A mixture of lipids (2.0 mg), consisting of egg phosphatidylcholine, phosphatidylglycerol, and MTP-PE in a 9:1:1 ratio, was dried under a stream of N2 and desiccated in a vacuum desiccator for 30 minutes. The lipids were then resuspended in injection water (2 mL) with sterile PBS (10 uL). The suspension was vortexed briefly, then sonicated for 15 minutes, providing a translucent liposome suspension. The suspension was passed through a 0.2 u filter for sterilization. Next, rgD-1 (120 ug) was added to the suspension (osmolar equivalent to 0.1 PBS), and the mixture frozen and lyophilized in a Vitus overnight.
Before administration, the lyophilized material was rehydrated in injection water (0.2 mL), vortexed, and allowed to stand at room temperature for 15 minutes. Then, 1.8 mL of injection grade saline is added, providing enough suspension for 4 doses of 0.5 mL (30 ug rgD-1).
Female Hartley strain Guinea pigs (400-500 g, Trents, Modesto Calif.) were divided into groups and administered 8.0 ug rgD-1 in one of the following vehicles:
A. Complete Freund's adjuvant (Sigma Chemical Co.) emulsified with PBS (boosted with incomplete Freund's adjuvant (Sigma Chemical Co.) in PBS (100 uL)
B. 10% alum, 100 mM "TRIZMA" hydrochloride (Tris[hydroxymethyl]aminomethane hydrochloride, Sigma Chemical Co.) (100 uL)
C. 50 ug Thr-MDP, 2.5% "PLURONIC L-121" (polyoxypropylene-polyoxyethylene block polymer, Syntex) 5% squalane, PBS (100 uL)
D. 50 ug Nor-MDP, 40% squalene, 10% ARLACEL A (mannide monooleate, Sigma Chemical Co.), PBS (100 uL)
E. 50 ug MTP-PE, 12.5 mg liposomes (7:3 phosphatidylcholine:phosphatidylserine), PBS (175 uL)
F. 50 ug MTP-PE, 4% squalene, 0.008% "TWEEN 80" (polyoxyethylenesorbitan monooleate, Sigma Chemical Co.), PBS (200 uL)
G. 50 ug MTP-PE, 40% squalene, 10% ARLACEL A (mannide monooleate, Sigma Chemical Co.) PBS (200 uL)
H. 62.5 ug RIBI (RIBI Vaccine Supply Co.) components, 2% squalene-Tween®80, PBS (250 uL).
All vehicles were administered in the hind footpad. Three administrations were performed at week 0, week 3, and week 6. The animals were bled for antibody determination on weeks 7 and 11.
Antibodies in the Guinea pig serum specific for rgD-1 were measured using an ELISA, using rgD-1 as the coating antigen. Antigen was diluted to a final concentration of 5 ug per well with PBS, and was adsorbed to the wells of a 96-well polyvinylchloride microtiter plate (Dynatech, Alexandria Va.). The plates were washed three times with PBS supplemented with 1% rabbit serum and 0.3% "TWEEN 20" (polyoxyethylenesorbitan monolaurate, Sigma Chemical Co.). Test sera were diluted serially with five-fold dilutions in the wash buffer, and incubated in the antigen-coated wells. All incubations were done for one hour, at 50 uL per well. The plates were again washed, and horseradish peroxidase-conjugated rabbit anti-guinea pig immunoglobulin (Boehringer Mannheim Biochemical, Indianapolis, Ind.) diluted 1:200 in wash buffer was added. Plates were washed with PBS and developed with 1 mg/mL "ABTS" (2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid), Boeringer Mannheim) (Boehringer Mannheim) in 0.1 M citric acid, pH 4.0, with 0.003% H2 O2. The color reaction was stopped after five minutes by the addition of 50 uL of 10% sodium dodecyl sulfate (SDS). The reported titer corresponds to the reciprocal of the dilution which produced an absorption at 414 nm equal to 50% of the maximum absorbance value of each serum.
The results were as follows:
______________________________________First TestGroup N Titer1 % CFA Titer2 % CFA______________________________________A 5 15,066 100 16,834 100 ±2,878 ±5,527B 4 1353 9 1118 7 ±318 ±211C 6 9641 64 7271 43 +1696 +1524______________________________________ N is the number of animals per group Titer1 is the Ab titer at week 7 Titer2 is the Ab titer at week 11 % CFA compares B, C titer vs group A (CFA)Group N Titer1 % CFA Titer2 % CFA______________________________________Second TestA 6 13,962 100 8,298 100 ±2,307 ±896D 6 10,387 74 5,759 69 ±2,964 +920E 6 9,898 72 2,653 32 ±1,161 ±457Third TestA 4 13,470 100 12,906 100 ±4,218 ±2,105H 6 13,994 104 7,833 61 ±4,157 ±3,946______________________________________Fourth TestGroup N Titer1 % Ribi Titer2 % CFA______________________________________H 4 8,000 100 1,318 100 ±1,271 ±310F 5 17,072 213 2,455 186 ±3,457 ±535G 4 14,731 184 1,181 90 ±3,860 ±433______________________________________
These results indicate that rgD-1 is immunoreactive when delivered in an appropriate vehicle.
Hartley strain female guinea pigs (250-300 g, EZH caviary) were divided into treatment groups as follows:
A. placebo (PBS), administered IM in hind leg
B. 12 ug HSV-1 rgD BIOCINE (HSV-gD-1 vaccine formula from Chiron Corp.), administered IM in hind leg
C. 0.2 mL liposomes (no antigen), administered by intra-cardial puncture (with anesthesia)
D. 12 ug rgD-1 in 0.2 mL liposomes, (administered by intra-cardial puncture with anesthesia)
Animals were challenged with 104 pfu HSV-2 administered intravaginally with a ball-tipped needle on day 0, and treated with one of the above formulations on days 14 and 28. Animals were bled on days 0, 14, 28, and 42.
Peripheral blood lymphocytes were isolated from guinea pig blood by "FICOLL" (a nonionic synthetic polymer of sucrose, Sigma Chemical Co.) gradient centrifugation. After washing the cells with tissue culture fluid, 2×105 cells were suspended with antigen and allowed to grow for 6 days. Triplicate samples were then pulsed with 3 H-thymidine for 6 hours on the sixth day, and the cells harvested with a cell harvester. Stimulation with phytohemagglutin (PHA) was used as the positive control, and results expressed as 3 H-dT uptake Ag/mock Ag (mock Ag=supernatant from cell cultures not exposed to HSV). The results were as follows:
______________________________________Group N LPI28 % Pos N LPI42 % Pos______________________________________A 10 3.5 ± 1.7 60% 10 4.5 ± 2.9 60%B 10 3.6 ± 3.2 40% 10 4.6 ± 3.2 70%C 11 10.4 ± 13.0 73% 10 4.9 ± 4.7 60%D 6 8.4 ± 2.8 100% 5 6.9 ± 2.8 100%______________________________________ N = number of animals per group LPI28 and LPI42 are the lymphocyte proliferation indices at 28 and 42 days, calculated as the ratio of 3 HdT incorporation for test group/3 HdT for mock antigen, reported as average and standard deviation. % Pos = % of animals in group with LPI ≧ 3 (positive CMI response)
The results demonstrate substantial increase in thymocyte proliferation, which is known to correlate with cell mediated immunity.
The animals were observed daily beginning with day 15, and any lesions graded on a 1-4 scale. The lesions are scored as follows: 1=no apparent lesion, 2=inflammation with vesicle formation, 3=severe inflammation with vesicles forming extended plaques, 4=tissue necrosis. The scores were recorded as averages for each group. The results are shown in FIG. 1, showing group A (open squares), group B (filled squares), group C (open circles) and group D (filled circles). The results indicate that the compositions of the invention are effective for substantial amelioration of symptoms from severe HSV-2 infection.
Animals were also observed daily for recurrences of HSV outbreaks. The cumulative total is plotted in FIG. 2, showing group A (open squares), group B (filled squares), group C (open circles) and group D (filled circles). The results indicate that the compositions of the invention substantially reduce the rate of recurrence.
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|US20060024393 *||Aug 12, 2005||Feb 2, 2006||Squires Meryl J||Antimicrobial prevention and treatment of human immunodeficiency virus and other infectious diseases|
|US20080274128 *||Jun 13, 2002||Nov 6, 2008||Rothman James E||Heat shock protein-based vaccines and immunotherapies|
|US20090098162 *||Dec 27, 2007||Apr 16, 2009||Harvey Friedman||Herpes simplex virus combined subunit vaccines and methods of use thereof|
|US20090191288 *||Oct 9, 2008||Jul 30, 2009||Squires Meryl J||Composition to Treat Herpes, Pseudomonas, Staph, Hepatitis and Other Infectious Diseases|
|US20100303935 *||May 29, 2009||Dec 2, 2010||Squires Meryl J||Medicinal Composition|
|US20100330112 *||May 24, 2010||Dec 30, 2010||Genocea Biosciences, Inc.||Vaccines against herpes simplex virus type 2: compositions and methods for eliciting an immune response|
|US20110177125 *||Sep 7, 2007||Jul 21, 2011||Harvey Friedman||Hsv-1 and hsv-2 vaccines and methods of use thereof|
|US20160256515 *||Oct 1, 2014||Sep 8, 2016||The Johns Hopkins University||Compositions and methods for prediction and treatment of human cytomegalovirus infections|
|US20160317649 *||May 17, 2016||Nov 3, 2016||Sydney West Area Health Service||Epitopes of herpes simplex virus|
|EP0603671A2 *||Dec 10, 1993||Jun 29, 1994||BEHRINGWERKE Aktiengesellschaft||Process of reactivating purified membrane proteins|
|EP0603671A3 *||Dec 10, 1993||Feb 22, 1995||Behringwerke Ag||Process of reactivating purified membrane proteins.|
|WO1997034583A1 *||Mar 24, 1997||Sep 25, 1997||Biosphere Technology||Method for inducing a systemic immune response to an antigen|
|U.S. Classification||424/196.11, 424/452, 424/231.1, 424/85.5, 424/279.1, 424/812, 424/283.1, 514/4.2, 514/20.9|
|International Classification||A61K39/245, A61K9/127|
|Cooperative Classification||A61K39/12, A61K2039/5555, A61K2039/54, C12N2710/16634, A61K2039/55555, A61K2039/55505, A61K2039/55566, Y10S424/812, A61K9/1271, A61K39/245|
|European Classification||A61K39/245, A61K9/127B|
|Oct 12, 1993||CC||Certificate of correction|
|Mar 21, 1996||FPAY||Fee payment|
Year of fee payment: 4
|Mar 21, 2000||FPAY||Fee payment|
Year of fee payment: 8
|Mar 22, 2004||FPAY||Fee payment|
Year of fee payment: 12

domain: biology


When you possess a new invention tip, it is essential to carry out some investigation. This will definitely consist of asking possible individuals what they think of your product. It is actually also a great idea to take a look at your competitions.
Be sure to work with a reliable design/invention organization, as well as possess them authorize an NDA. This will definitely safeguard your trademark and also avoid all of them from swiping your tip.
When you have a brand-new creation tip, it can be actually difficult to recognize just how to get begun. It is additionally essential to produce certain that your item will definitely meet the demands of your target audience.
One more crucial step is actually to record your development tip. The business InventHelp gives brand new makers a variation of services, consisting of development chronicling and also model creation. How much does InventHelp cost?
In order to come up along with really good brand new invention suggestions, you need to concentrate on an area of experience. It is crucial to keep in mind that even the littlest problems can lead to excellent developments.
Developers typically generate tips for items that may aid to create a distinction in our lives. While this can be a wonderful trait, it is vital for brand-new inventors to receive the correct help so they can easily take their innovation suggestion to the upcoming degree. Getting this assistance can aid to create the whole development procedure much easier, and it can easily additionally make the brand new creator experience additional self-assured in their potentials.
One of the largest obstacles for brand-new makers is acquiring services to take an interest in their creation. However, this is something that InventHelp can assist with. They possess a data bank of companies that agree to review the tips of brand new developers, as well as this can be a large advantage for those who wish to transform their concept in to a genuine product.
Additionally, InventHelp may support with various other methods that are essential to the development adventure, featuring obtaining patent security in position as well as making a prototype These are factors that several brand-new makers do not deal with, as well as they can easily possess a substantial influence on the excellence of their innovation.
Lots of folks additionally wish to know if the specialists at InventHelp will be able to provide their viewpoint on their creation tip, but this is something that they may not do ethically. This is actually given that it will be unfair for them to compete with their consumers, and also it is additionally illegal for all of them to perform so.
Getting a model.
Obtaining a model for your new innovation is a crucial step in the procedure of transforming an idea into a product. It enables you to obtain user feedback, excellent your style, as well as create new patentable features.
InventHelp may help you with the prototyping procedure, as well as their group of pros is extremely skilled in the field. They can aid you with many processes, featuring patent defense and model development. These are very important aspects of the development method, and InventHelp has actually been actually providing these companies for a long times.
A prototype is a rugged physical variation of an innovation. If your development is unreasonable or costly to model, take into consideration using a computer-animated virtual model.
Developing a brand new product is a lengthy quest, and it takes a while to get to the last. Hurrying the growth process can result in a defective item or even a tip that is never know. A productive product is actually the outcome of a great deal of effort as well as resolve. How to patent an invention idea?
Obtaining a license
Receiving a patent is a significant come in the innovation process, as well as it can secure your intellectual property. It provides you the right to stop others coming from utilizing your innovation without permission, and also it enables you to earn earnings for your idea. It can easily additionally be utilized to prevent competitions coming from duplicating your service or product, and you may sue them for significant volumes of loan.
InventHelp can support brand-new inventors along with several processes, consisting of helping them get their creations patented. They can also help them create models and also discover entrepreneurs. It is vital to locate a reliable provider, nonetheless, as there are actually several hoaxes around. Be wary of providers that guarantee warranties or even deal point of views on your innovation.
It is likewise an excellent suggestion to do some investigation before you go after license security. You need to guarantee that your invention is actually one-of-a-kind and also it has a market. Furthermore, you need to make sure that the expenses of production and also circulation prove out. If you possess a great development, but it is actually as well costly to generate, you could would like to think about licensing your idea to a growing business.
To defend your innovation, you need to have to declare a provisional patent. This is actually a less costly and faster substitute to the full patent process, and also it uses the same lawful defense. You can get a conditional patent online through the USPTO internet site, but make certain to go through the directions very carefully.
When you possess a new innovation idea, it’s vital to perform some research study. When you possess a brand-new innovation concept, it can be complicated to know just how to obtain begun. In order to come up with really good new creation ideas, you need to have to center on an area of expertise. While this may be actually a fantastic thing, it is crucial for new developers to obtain the ideal assistance so they can easily take their development suggestion to the following degree. Receiving a model for your brand new development is actually a necessary measure in the process of turning a suggestion right into an item.


text:
Frequently Asked Questions
Healthy eating is not cheap! Healthful foods are not only expensive they are also inaccessible to the poor. The majority of places known as food deserts in Oklahoma have high rates of diabetes, heart disease, and certain types of cancers. Many people are uneducated on how to eat right and how food affects the body and mind. A Table in the Wilderness is embarking on a long-term effort to educate Oklahomans on healthful living, providing plant-based foods to people with limited access to it, and reaching people with the everlasting Gospel.

domain: food


text:
The Australian Taxation Office (ATO) has told Australians that many of its services are back online following a weekend-long marathon effort working with Hewlett Packard Enterprise (HPE) engineers to replace failed hardware and restore systems.
The agency said early on 6 February that a number of core services, including the Tax Agent, Business and BAS Agent Portals, ATO Online services, and Standard Business Reporting (SBR) services were back up and running, with other services expected to be brought online “shortly”.
“Our clients may experience some slowness as further work is undertaken to improve the overall performance of our systems,” the ATO said in a statement.
“Our focus will now turn to building system resilience to best ensure the stability of our services to the community.”
The latest update on the ATO’s long-running efforts to restore its systems following a massive outage caused by storage hardware failure on 12 December 2016 comes after days and nights of work that saw ATO staff join forces with HPE technicians to replace the storage hardware and get systems back up and running.
“Engineers from Hewlett Packard Enterprise and the ATO continued system restoration efforts overnight and this morning,” the ATO said on 5 February.
“Good progress has been made as we work towards having services available for clients on Monday. This will however be subject to ongoing testing of the integrity and stability of the system.
“Restoration efforts are progressing well and engineers are now undertaking technical verification of our systems,” the agency later said. “This will continue throughout the night with a view to services being available tomorrow.
“Work has also been completed to ensure we have appropriate contingencies in place which is giving us increasing confidence that we will at least have our core client services available tomorrow [6 February],” it said.
The ATO flagged on 4 February that HPE would work over the weekend to restore systems, with support from the agency’s own technicians. At the time, it indicated that it did not expect services to be available until after 5 February.
“While we are working as quickly as we can, we cannot make services available to the community until the integrity of the system is confirmed,” the agency said.
The agency also revealed that it had commissioned a new Storage Area Network (SAN) to “provide better services in the future,” saying that the new hardware had arrived at ATO premises.
“This new system will contain vast amounts of ATO data and it is currently being configured to provide more reliability and stability,” the ATO said at the time.
The nature and scope of the ATO’s SAN means that this process of replacing the affected hardware will take some time.”
Join the CIO Australia group on LinkedIn. The group is open to CIOs, IT Directors, COOs, CTOs and senior IT managers.

domain: news


text:
The Shevchenko’s are in town! UFC fighter Valentina Shevchenko (14-2) has landed in Lebanon alongside her sister Antonina Shevchenko prior to Antonina’s fight at Phoenix 2 event this weekend.
Antonina Shevchenko will square off against Muay Thai World and European Champion Isa Tidblad Keskikangas or the prestigious Female WMC World Title belt for the first time in the Middle East. She (Antonina) is a 7-time Muay Thai champion and 3 times World IFMA Muay Thai Champion with a 38-1 kickboxing/Muay Thai record.
Check out this Instagram video of Antonina doing her workouts, while her sister gives us a peak of the scenic gardens at the Country Lodge resort.
Proud to represent and promote female fights in the Middle East, the Kyrgyztani is ready for her match-up this Saturday in Lebanon’s biggest promotion, Phoenix Fighting Championship.

domain: geography


text:
Every type of investor can find something profitable in real estate. Commercial real estate funds are becoming more visible on the market as small-time investors increasingly find appeal in the prospect of receiving a steady passive income from safe real estate assets. For these investors who do not have a portfolio worth a crore, the upcoming year may bring a lot more alternatives. Read on to know more about it.
Predictions for the Real Estate Sector in 2023
The Indian real estate market has been faithful to investor expectations through the many ups and downs in the last few years. It is time-tested and widely thought to be impervious to market volatility. Many analysts feel that positive expectations and upward momentum will continue in the upcoming year because they have demonstrated their ability to sustain themselves. Young investors might be quick to label real estate as “slow” or “boring,” but this is unlikely to be the case.
The total Indian economy is projected to grow by 8–9% in 2023, with increased job creation anticipated as well as a rebound from the recent stock market downturn. These are all indicators that can only be interpreted as good. All of these would ultimately lead to a rise in housing demand as more individuals entered the housing market, which is crucial for the health of the real estate industry. In addition to important policy efforts like “Housing for all,” we are aware of the government’s actual planning and implementation of infrastructure mega-projects like new airports, metros, and motorways. Such factors promote both quantitative and qualitative growth in real estate assets. The recent government rollout of numerous tax-related and other incentives aimed at prospective home buyers as well as the streamlining of the required bureaucratic work are additional factors that experts believe could have a significant positive impact on the real estate industry.
Low-interest rates are also thought to have a significant positive impact on the real estate industry. The tier-II and -III real estate markets, which are frequently disregarded and ignored, stand to gain the most from these encouraging trends, and it is very possible that they will bring substantial returns for investors.
As large IT businesses return to offline work, commercial real estate, one of the most desirable assets in the real estate game, is also expected to experience excellent returns. Many retailers who hesitated to create actual stores because of the pandemic are now progressively changing their minds. Additionally, e-commerce companies are competing with one another to grow as quickly as possible by increasing their logistics and warehousing operations.
The housing industry, which fuels economic expansion and supports the whole real estate sector, is about to see one of its most outstanding and possibly finest years ever. Residential sales have increased dramatically since the Covid-19 period. Prices are predicted to climb by about 7.5% nationwide, according to Reuters and a variety of other economic indicators point to a good improvement for the real estate sector.
It can be expected that the real estate sector is going to show positive results in 2023. However, it would be wrong to say that it won’t come with any setbacks as increasing mortgage rates, inflation, and building material shortages might pose challenges but if these issues remain confined and limited in extent, no major threat or problem can be anticipated. The combination of a developing economy, a return to pre-pandemic working conditions, and different government efforts are fostering a climate that will be very favorable for the real estate market’s success in the years to come.
Achyut Group is a trusted brand due to its ethical values among its customer, vendors, and competitors. We believe in creating environment-friendly green buildings by conserving energy, water, and wood. The promoters of Achyut Group combine over 20+ years of experience in the construction legal, finance, and marketing sectors.


text:
I love otters. They’re just so adorable. But I never knew they liked Christmas! Okay, okay, they really just like treats made with minced clams, but it still makes for a great video. Enjoy (and Merry Christmas)!
Common threads2020 Benedict Cumberbatch board games books buy borrow bin cancer creative writing prompts cute cute cute depression and anxiety Discworld Doctor Who Douglas Adams family fashion fiction writing flash fiction friends Funko Hobbit/LOTR hurricanes illness joy and laughter Katrina life Lovecraft Marvel Cinematic Universe music Nathan Fillion novel otters reading reading challenge Richard Armitage Sherlock short stories story submissions stress TableTop Terry Pratchett Tom Hiddleston wildlife writing exercises writing workshop

domain: artistic


Bluetooth 5.0 Audio Module TPA3116D2 Stereo Power Digital HIFI 2.0 power Amplifier Board 2x50W
—CHIP: TPA3116D2 (with AM interference suppression function), 50W*2 output power With filter
—Work Voltage:DC 5V-27V Above 2A is recommended
—Communication distance:15meter(Without obstacles)
— Tip: adequate output power is required when the audio input is sufficient and the power supply voltage/current is sufficient, the relative power will be higher. Different impedance horns will have different output power.In the case of sufficient voltage and current, the larger the impedance horn, the smaller the relative sound power, please pay attention!
—TWO AUDIO INPUT WAYS:User Can Directly Connect with Wireless Bluetooth 5.0, or Choose the Headphone (Line in) Way
—PROTECTIVE FUNCTION:This BT Amp Is Designed with Protective Case, Reverse Connection protection, Over-voltage Protection, Under-voltage Protection, Over-heat Protection, Short-circuit Protection


text:
"Soulmates" Carol (Melissa McBride) and Daryl (Norman Reedus) "have a very big storyline together" in The Walking Dead's tenth season, says showrunner Angela Kang, who adds the longtime best friends will be on the frontlines in the renewed fight against Alpha (Samantha Morton) and her Whisperers.
"We'll see a lot of the Whisperers conflict through the eyes of these two as they are at times on parallel tracks, at times on divergent tracks," Kang told TVLine. Though Carol gives Daryl a rough time for calling her his "best friend" in a moment shared between the two in the Season 10 premiere, "they are best friends," Kang said.
"I think they are soulmates, whether or not their relationship ever goes into romantic territory. They are tied to each other in this world and have been through so much together," Kang continued. "They love each other, and they trust each other. They care what happens to the other. And yet that can be very complicated when there's so much trauma and this bigger thing that's taking over the communities."
In Season 10, Carol is still healing from the murder of son Henry (Matt Lintz) — one of nearly a dozen victims claimed by Alpha — a loss that drove her to a "pirate's" life at sea. When reunited with Daryl, she learns he strictly adheres to Alpha's warning not to cross into her territory — as he tells it, there's "no reason to start sh-t if we don't gotta."
"Melissa's lovely, but Carol has been a real pain in my butt for Daryl this season, I will say," Reedus previously told Entertainment Tonight. "I love working with Melissa, she's dynamite. We have such a good rapport and we're good friends, and we have fun."
When asked if the new romantic development between Michonne (Danai Gurira) and Carol's ex-husband Ezekiel (Khary Payton) meant there was a chance for the "Caryl" pairing to progress beyond platonic, Reedus answered, "I don't know if it's this year, because like I say, she's a little bit cray cray this year. But I mean, never say never. I don't know. She's a problem this year, you know what I'm saying?"
Despite Carol's trouble stirring, there's "just some really beautiful stuff" between Carol and Daryl in McBride and Reedus' tenth season together.
"I love the two of them together. They're so good together," Kang recently told EW. "There are certain character pairings because we've been following them for so long where they're in a scene and you immediately feel the history between those characters. That's one of the things that's been really fun for us on the writing and producing side to see. Anytime you put those two in a scene, you immediately feel all the weight of the things that they've both been through as individuals and as a pairing over the years. There's just some really beautiful stuff between them."0comments

domain: entertainment


text:
Now, the good Frater Olen Rush recently published a transcript from a visionary experience back in 1888 by one Frater Vota Vita Mea, that is Thomas Henry Pattison which is listed as No. 8 of the Horus Temple No. 5 membership roll, admitted as a 5°=6° by march 1888.
This letter has previously been published in full by R.A. Gilbert in his The Golden Dawn Scrapbook, pages 37-39. The transcript reeds in full thus (retaining comments by Mr. Gilbert within square brackets):
Copy of Communication from the Inner Ring, made to Fra. Vota Vita Mea, given in his letter of June 20, 1888 to Fra Sapere Aude:
The Crowned One gave to Fras. V.V.M. and H. [presumably F.D. Harrison] much information concerning the natural creation and the following symbol was the one he adopted to illustrate primary creation or the union of ﬁrst causes:
After this Fra. H. was conducted by an Eagle-headed messenger into the presence of the Angel R + [Raphael] (whom I, S'Rioghail Mo Dhream, know by the motto L[ux] e T[enebris]) He said:Now, people has of course noted the particular mention by S.L. MacGregor Mathers of Lux e Tenebris, the motto that MacGregor Mathers re-used for his later in the flesh Secret Chief which he met in Paris in 1892. Some have therefore speculated that MacGregor Mathers started out with a belief of solely astral or angelic powers as the source of the Third Order, and that he later on changed his beliefs into the concept of continental Rosicrucian initiates.
‘I wish to speak to you of the G.D. The prime mover of it must exercise the keenest possible care, and must not accept people into its secrets who are not Occult Students, they all ought to have some occult development which has been the outcome of their own research; otherwise much Evil Magic will be the result. Much good can come out of it if rightly directed, but one Evil One can upset the Harmony of the whole, so that its efforts would be neutral for a lengthy period. The Idea in view must be not to establish an Order; but to gather together those men who are an Order In themselves. Understand that the G.D. is MY Order and that I am and must be accountable for it. The last one initiated [Dr T.W. Coffin, who left the Order within a year having remained in the grade of Neophyte] must be considered and allowed to wait before giving information; 2 or 3 others also are not of much use, and others are likely to drop out. You must take every care not to initiate any who have no occult tendency in a degree developed. Convey my message to those who can carry out my advice.
‘You must consider yourselves highly favoured indeed for being allowed to come in contact with the one (the Crowned One) you did tonight.
‘Also, I would caution and advise you to be very careful in the use of Divine Names, and all symbols should (be) correctly traced. I have seen this lady in Switzerland whom you have been trying to release. I conﬁrm the work you have attempted. But you have failed to fully carry out your intention. The failure is due to the want of correctness in carrying out your Ritual. When the Divine Names are used they are made operative by the emotion of the Soul; if uttered by a cold soul-starved man they are of no use. Put your soul-power into them, and let your whole being speak them; then they do become Operative; and the ﬁnal Issue of this lady in Switzerland is left open at present for your instruction. You have discovered and commanded the assailants, but you have not yet subdued them; although she will be very carefully watched now: You must make your promise of protection a fact, and establish it as a permanent operation. I shall see you again as to this.’
He also said; - ‘The G.D. will soon ﬁnd the prime movers plenty to do; they will have more applicants than they can take; and if not careful will get beyond their control.’
He then said to Fra. H, ‘You can go’; and the Eagle-headed Messenger conducted him back.
For me this seems plausible, and even more plausible that he believed in both. I personally believe that MacGregor Mathers used the Lux e Tenebris phrase as a generic motto for whatever Secret Chief or source of inspiration he had come into contact with or believe in at a certain point. After the meeting with his physical Secret Chief in Paris he started to apply that motto onto him. Later on there started to spread a false rumour that the identity of L.e.T. was a Dr. Thiessen of Liege, who supposedly died in 1909. However, it’s a well known fact amongst initiates that the L.e.T. motto is a blind, and that the real motto and true identity of MacGregor Mathers’ continental Secret Chief is still secret to this day.
However, one reconstructionist and history revisionist author has even suggested that MacGregor Mathers must have referred to the infamous Madame Horos as his Secret Chief when he announced that he had been visited by them in the flesh. But in his usual manner this author hasn’t made his homework sufficiently enough and completely either missed or ignored the fact that MacGregor Mathers’ in the flesh contact with Madame Horos was established in early 1900, while he made his first official and written claims of physical contact with his Secret Chiefs back in 1896, that is four years prior to him taking in Madame Horos and her husband as guests at his Paris house. He also totally misses the fact that MacGregor Mathers in a weak moment (probably his weakest) believed Madame Horus to be fräulein Sprengel, who used the motto of Sapiens Dominabitur Astris, not that of Lux E Tenebris.
Now let me present the facts, not any fantasies, and again quote MacGregor Mathers’ manifesto concerning the Secret Chiefs in full:
The Manifesto of G.H. Frater Deo Duce Comite Ferro 7° = 4°, Adeptus Exemptus, Chief Adept and Ambassador of those Secret and Unknown Magi who are the concealed Rulers of the Wisdom of the True Rosicrucian Magic of Light:It is my belief that this manifesto should be treated as an official order document and statement on the nature of the Secret Chiefs and the Third Order, at the time period of MacGregor Mathers’ most frequent encounters with them. I would also in this instance want to quote W. B. Yeats from his Autobiographies, written in 1926. Here Yeats recalls a conversation with MacGregor Mathers concerning the the Secret Chiefs:
The Theorici Adepti Minores of the Order R∴ R∴ et A∴ C∴
Notice: This Manifesto is to be placed in the hands of each Theoricus Adeptus Minor upon his or her attainment of that Grade. After he or she has carefully read the same; he or she must send a written Statement of voluntary submission in all points regarding the Orders of the G∴D∴ in the Outer and the R∴R∴ et A∴C∴ to G.H. Frater Deo Duce Comite Ferro before being permitted to receive any further instruction. Unless he or she is prepared to do this, he or she must either Resign from the Order, or elect to remain a Zelator Adeptus Minor only. And he or she hereby undertakes to refrain from stirring up any strife or schism hereon in the First and Second Orders.
It is necessary that you who have now attained the Grade of Theoricus Adeptus Minor after having passed through the numerous examinations on the Secret Knowledge of the Zelator Adeptus Minor Grade of the Second Order should now understand how that wonderful system of Occult Wisdom has been obtained for you.
Prior to the establishment of the Vault of the Adepts in Britannia (the First Order of the G∴D∴ in the Outer being therein actively working) it was found absolutely and imperatively necessary that there should be some eminent Member especially chosen to act as the link between the Secret Chiefs and the more external forms of the Order. It was requisite that such Member should be me, who, while having the necessary and peculiar educational basis of critical and profound Occult Archaeological Knowledge should at the same time not only be ready and willing to devote himself in every sense to a blind and unreasoning obedience to those Secret Chiefs: - to pledge himself for the fidelity of those to whom this Wisdom was to be communicated: - to be one who would shrink neither from danger physical, astral, or spiritual, from privation or hardship, nor from terrible personal and psychic responsibility; one who, while receiving for transmission the Hidden Wisdom of the Rosy Cross, should be willing to pledge himself under the severest penalties possible, that the Order should be worked in conformity with the principles laid down by those Secret Chiefs not only for the present time but in the future also: - and who should further possess an Iron Will unable to be broken by any unlooked for opposition that might arise in the carrying out of these duties: - he must further pledge himself to obey in everything the commands of the aforesaid Secret Chiefs 'perinde ac cadaver' body and soul without question and without argument whether their commands related to: - Magical Action in the External World; or to Psychic Action in other Worlds or planes, whether Angelic, Spiritual, or Demonic; or to the Inner Administration of the Order to which so tremendous a Knowledge was to be communicated; and that he must further be prepared to abide in any country; to undertake any journey at a moment's notice, or to confront the chances of death, pestilence or elemental upheaval, if called upon in the course of fulfilling their demands to do so; that he would further undertake, whatever might occur, never to lose faith in the Chiefs of the Order, and to keep his body in such a condition of physical health and especially of vital energy, that the ordinary chances of corporal illness and exhaustion should not be permitted to become any bar to his constant efforts and exertions: - all this and yet further conditions were insisted upon as the only pledges under which this Divine Wisdom was to be permitted to be given out: - and these had to be confirmed by the most terrible Obligations.
I, MacGregor Mathers, 'S Rioghail Mo Dhream 5° = 6°, Deo Duce Comite Ferro 7° = 4°, was the Frater selected for this Work: whom you know as the Chief Adept of the Second Order under the title of Deo Duce Comite Ferro which I had taken upon me.
At my urgent request Soror Vestigia Nulla Retrorsum [i.e. Moina Mathers] was allowed to be associated with me in this labour, but only on condition of pledging herself in the same manner, though in a less degree.
With all this it was insisted that I, the aforesaid Frater, and in a less degree the aforesaid Soror were to be held responsible for any action undertaken thro' misapprehension of the instructions of the Chiefs, no matter in what manner those Instructions were to be conveyed.
That yet further it must not be expected that these pledges should confer upon me any right to expect abnormal material support or assistance. The conditions in question, being only those on which I was to be allowed so great an honour as to be the Recipient of the Knowledge for transmission to the Order; and also that in all this my obedience was not to be the passive one of an unreasoning Machine, but the active one of the intelligent and voluntary Ministrant of the Magic of the Eternal Gods.
These preliminary conditions having been solemnly undertaken by us; then, and only then was I able to proceed to the attainment of the Knowledge of the full 5° = 6° Ritual and of the Obligation thereof; and the establishment of the Vault of the Adepts; my quitting England being a necessary preliminary thereto.
The working of the Second Order having been thus initiated I was enabled to proceed to the acquirement of the Wisdom of the Zelator Adeptus Minor Grade for transmission to you: - a work, the enormous strain and labour of which it is impossible for me to exaggerate. For, you must not think that the obtaining of this Knowledge of the Second Order for you has been merely and simply the somewhat commonplace labour of translating a heap of unclassified MSS ready placed in my hands for that purpose. This might indeed be difficult and fatiguing, but it would be the merest child's play compared with the Herculean task that I have been called upon to execute.
Concerning the Secret Chiefs of the Order, to whom I make reference and from whom I have received the Wisdom of the Second Order which I have communicated to you, I can tell you nothing.
I do not even know their earthly names.
I know them only by certain secret mottoes.
I have but very rarely seen them in the physical body; and on such rare occasions the rendezvous was made astrally by them at the time and place which had been astrally appointed beforehand.
For my part I believe them to be human and living upon this earth; but possessing terrible superhuman powers.
When such rendezvous has been in a much frequented place, there has been nothing in their personal appearance and dress to mark them out as differing in any way from ordinary people except the appearance and sensation of transcendent health and physical vigour (whether they seemed persons in youth or age) which was their invariable accompaniment; in other words, the physical appearance which the possession of the Elixir of Life has traditionally supposed to confer!
On the other hand, when the rendezvous has been in a place free from easy access by the Outer World they have usually been in symbolic robes and insignia.
But my physical intercourse with them on these rare occasions, has shewn me how difficult it is for a mortal, even though advanced in Occultism, to support the actual presence of an Adept in the Physical Body; and such meetings have never been granted to my own personal request, but only by their own special appointment; and usually only for some reason of special importance.
I do not mean that in such rare cases of physical converse with them that the effect produced on me was that intense physical exhaustion which follows depletion by magnetism; but, on the contrary, the sensation was that of being in contact with so terrible a force that I can only compare it to the continued effect of that usually experienced momentarily by a person close to whom a flash of lightning passes during a violent storm; coupled with a difficulty in respiration similar to the half-strangling effect produced by ether; and if such was the result produced in one, as tested as I have been in practical Occult Work, I cannot conceive a much less advanced Initiate being able to support such a strain even for five minutes, without Death ensuing.
Almost the whole of the Second Order Knowledge has been obtained by me from them in various ways; by clairvoyance, by Astral projection on their part and on mine-by the table, by the ring and disc, at times by a direct Voice audible to my external ear, and that of Vestigia, at times copied from books brought before me, I know not how, and which disappeared from my vision when the transcription was finished, at times by appointment Astrally at a certain place, till then unknown to me; and appointments made in the same manner and kept in the same manner as in the case of those rare occasions when I have met them by appointment in the physical body.
The strain of such labour has been, as you can conceive, enormous; in especial the obtaining of the Z ritual, which I thought would have killed me, or Vestigia or both, the nerve prostration after each reception being terrible from the strain of testing the correctness of every passage thus communicated; the nerve prostration alluded to being at times accompanied by profuse cold perspirations; and by severe loss of blood from the nose, mouth, and occasionally the ears.
You know the extreme and sustained attention and critical judgment requisite to obtain any reliable and truthful answer through the Table or the Ring and the Disc. Add to all this Ceremonies of Evocation, almost constant strife with opposing Demonic Forces endeavouring to stop the delivery and reception of the Wisdom; and the necessity of keeping the mind exalted towards the Higher Self; while at the same time exercising the critical Archaeological Knowledge and having to make the many references necessary to detect any misapprehension of meanings of passages in Latin, Greek, Hebrew, Chaldaic, Egyptian and what not; and you will only then have a faint idea of what my struggles have been. The only one among you who has known the fearful difficulties I have had to contend with, has been Sapere Aude [Westcott] and he has therefore well known that such work could not be done in a hurry and rapidly like mere mechanical transcription, or even like ordinary original composition.
But unless the Chiefs are willing to give me the Knowledge, I cannot obtain it for you: - neither will I give it to you unless I know that the Order is being worked conformably with their wishes and instructions.
What I discountenance and will check and punish whenever I find it in the Order is any attempt to criticise and interfere with the private life of Members of the Order; neither will I give the Wisdom of the Gods to those who endeavour to use it as a means of justifying intolerance, intermeddling, and malicious self conceit. The private Life of a Person is a matter between himself or herself, and his, or her God; and no person who has taken the obligation of 5° = 6°, and studied the same can be ignorant of its clauses and penalties.
The Temples of the Order are places for the performance of Sacred Ceremonies, and the petty criticisms and uncharitableness of social clubs and drawing rooms, should be rigidly banished from them. To invoke carelessly and inadequately the Divine White Brilliance and the Forms of the Eternal Gods while permitting your mind and lower personality to be filled with uncharitableness; towards your neighbour, self righteous pride, and trivial social considerations, is an abominable blasphemy; it is that taking the Name of God in vain which is a most pernicious sin; for by so doing you cannot touch the God, but instead and in his semblance arouse the Evil Antithesis.
This, I know, has been a sin among you during the last twelve months; and be sure that this must bring its reaction; and there is no surer road to this error than the encouragement of the feeling of the Pharisee: ‘God, I thank thee that I am not as other men are’. Such a formula, whether expressed or implied, means at once separation from that God who is universal.
Of what use are the Second Order Centres, if they are not places where the Gods and the Angelic Forces are invoked in Spirit and Truth; and where Mystic Powers have their abode, and where petty social gossip can find no place.
It is possible for you to be word perfect in all the knowledge of the Zelator Adeptus Minor Grade and to know all its Ceremonies by rote; and yet unless you can really and profoundly grasp their inner meaning, an uninitiated person who has a strong will, faith, reverence, self sacrifice, and perseverance, may be more truly a Magician than you.
Finally, the reading of this Manifesto will have made you comprehend the enormous amount of time and energy necessary to obtain the Wisdom of the Second Order. To this must be added the considerable amount of labour connected with the Order - my own work in the outer world - and with all this the imperative necessity of keeping my physical health and vital energy always up to full pitch; and at times having had the extra disadvantage of being compelled to earn my own living in the world.
I therefore expect you to aid me to the best of your ability by carrying out my wishes regarding the management of the Order and by abstaining to the utmost of your power from putting any extra hindrance in my way.
I have considered it advisable that you should have both had in your possession and carefully studied the whole knowledge of the Zelator Adeptus Minor Grade before receiving this Manifesto.
S. L. MacGregor Mathers. 'S Rioghail Mo Dhream
Deo Duce Comite Ferro 7° = 4°
87 Rue Mozart, Auteuil, Paris
October 29th, 1896
Once, when Mathers told me that he had met his Teachers in some great crowd, and only knew that they were phantoms by a shock that was like an electric shock to his heart, I asked him how he knew he was not deceived or hallucinated. He said, “I had been visited by one of them the other night, and I followed him out, and followed him down that little lane to the right. Presently I fell over the milk-boy, and the milk-boy got in a rage because he said that not only I but the man in front had fallen over him.” He, like all that I have known who have given themselves up to images, and to the speech of images, thought that when he had proved that an image could act independently of his mind, he had proved also that neither it, nor what it had spoken, had originated there.This account fully corroborates MacGregor Mathers’ beliefs as shown in the above quoted manifesto and fully refute Madame Horus as his Secret Chief, which is a proposperous and outrageous proposition in the first place. A further evidence of this is that it is clearly stated in the manifesto that the reason of MacGregor Mathers and his wife Moina to leave England and permanently moving to Paris was to come closer to his Secret Chiefs, such as L.e.T.; thus it reads:
I [was] able to proceed to the attainment of the Knowledge of the full 5° = 6° Ritual and of the Obligation thereof; and the establishment of the Vault of the Adepts; my quitting England being a necessary preliminary thereto.S.L. MacGregor Mathers’ statement must be placed in juxtaposition with what Moina Mathers, in her preface to her husband’s book The Kabbalah Unveiled, wrote in 1926 concerning this matter:
Three or four years later he was told by his Occult teachers to transfer his centre to Paris, where my husband and I lived for the rest of his life.There is no doubt in my mind that Moina clearly refers to S.L. MacGregor Mathers’ prime motivation to move to Paris as to facilitate his communication with his Secret Chiefs. If they were solely astral or angelical contacts, surely there wouldn't be any need of a physical relocation. These written statements, when compared together, for me clearly confirms that his Occult teachers were real and physical individuals.
It is fine by me that there is a variety in the Golden Dawn community. It is fine by me that we share different views regarding the Secret Chiefs and the Third Order, or even that some deny the very existence of the latter and regard it only to be a myth, etc. Each to his own. But what I cannot accept is that facts are being twisted around to serve a militant reconstructionist agenda.
I have watched this debate regarding the Secret Chiefs silently up until now. I honestly don’t care what others think about them or about others’ opinions about my beliefs. These days I’m not surprised at anything anymore; there are obviously no limits to the amount of delusions in the community. Without any connection with the tradition to restraint the imagination people have all sorts of personal opinions and are damn proud to present them all over the internet. The idioticy of the Golden Dawn community is starting to get so piled up it must be almost impossible to wade through it as a prospecting initiate. People may actually start to believe in these fantasies. That’s why I feel obliged to set the record straight.
This whole matter has made me ponder the sad and sordid state of the Golden Dawn community. It has dawned upon me for the last couple of days that it [i.e. the community] produces scholars rather than Adepts. Many today even equate them two. Since the dawn of the last revival of the Golden Dawn Tradition in the 1970’s, it has been a subject to historical research with scholars as Ellic Howe, Francis X. King and R.A. Gilbert. This has put a tone to the whole current. Adepts of the tradition has followed suit en masse.
Now, I do believe that scholarship is a good thing, but if taken moderately. To many initiates and students of the Golden Dawn have an overly detached and dispassionate attitude towards its Tradition. It is not a historical curiosity that can be reconstructed at will; it’s a living Tradition that wants to express itself according to its own rationale. Is has its own soul, it is an entity which acts as an intermediary between the Divine and the mundane. It is possible that the first quoted document (from V.V.M.) captures that notion and spirit. Read it again from this point of view.
Where are the feelings of awe and sacredness in relation to the Tradition? I don’t know about you, but I have had a strong and passionate love affair with the Golden Dawn. For me It is truly a mistress. A jealous mistress, yes, but one which also comforts me and makes me aspire to better myself as a human. I am never alone with the Golden Dawn. She is a infinite source of joy and inspiration. The richness of the Golden Dawn, of its written and living Tradition, is awe inspiring. It is a beautiful Tradition which has been passed on to us to be managed as its stewards, for the betterment of its initiates. As initiates we are her children. Some of her children have been more able than others to act as her vehicles for transmission and expression, which has developed or reformed the Tradition further. These are the most proficient Adepts and prominent teachers, who would naturally become the Chiefs.
Granted, the Golden Dawn is not a religion, and the Order is not a Church. Neither should it be a personality cult. But the Order is not an academic institute, nor is its teachings any science theory. The Golden Dawn is a spiritual path which can be equated with any spiritual path, also those as represented by different religions and Churches. The Order is a Holy institution. It is eternal. Its Halls are sacred as well as it rites; its Officers represent and channel divine powers. It is part of a long chain of vehicles which has transmitted a divine knowledge to aspiring initiates who search for Apotheosis and the attainment of the Philosopher’s Stone. These initiates are aware that by themselves they may produce some minor effects, but with the aid of the Order they become able to create vertible Wonders.
It is only if you approach the Temple with reverence and true spiritual love and compassion that you can gain that sought after illumination. Show deep respect for the Adepts that has walked these steps before you. Show deep respect for its former teachers who has presented the teachings of the Tradition for your own benefit. For your own benefit, mind you!
Would any eastern brother scorn and ridicule its Master or teacher? Would a tantrika downplay on the achievements of previous Masters and teachers of his kaula (spiritual family)? Would a Buddhist show disrespect or spite towards the Boddishatvas? Would a Qabalist badmouth any of its long chain of Masters, or Tzadiks, such as Isaac Luria, Sabbathai Zvi, Nathan of Gaza or Jacob Frank? Why do you think a traditionalist Qabalist makes homage to these individuals whenever he mentions their name, in the same manner as a muslim gives honour to the prophet? Do you believe a Martinist would slander Martinez de Pasqually or Louis Claude de Saint-Martin? No? Why do you think? Because they were perfect Masters? Think again.
Now, I’m not admonishing anyone to feel reverence to S.L. MacGregor Mathers or to place him upon a pedestal. He is not a demi-god; he wasn’t a saint. What I’m saying is that we as initiates and Adepts should feel respect and gratitude to him, and other past teachers of our Sacred Order and Tradition, such as W.W. Westcott, J.W. Brodie-Innes or R.W. Felkin, and even Paul Foster Case and Dion Fortune. I happen to believe that MacGregor Mathers wasn’t that monster as some has tried to make us believe. I happen to believe that he was a good man and a great teacher, a dedicated person who did his very best to make each and everyone of us, who are heirs to his achievements, to gain communion with our God. However, this is not at all about canonisation.
Being initiates we belong to a family with ancestral roots that goes back to antiquity. The Golden Dawn is but the latest form that this entity or ancient egregore has taken in our modern era. We as initiates are part of that egregore. We have the duty to pass on the tourch, which has been passed on to us from our elder brethren and sisters, some of who are historically known and others are not. When we receive the torch we must aspire to leave it in a better condition that it was previously, when the time finally arrives to pass on the torch to further generations. We do not better our Tradition and perfect our Order’s teachings by badmouthing our predecessors, who did the very best that they could manage considering their resources and individual abilities. We do it by setting our own example by our own hard work in perfecting our own base nature. Through our hard work may we come to understand not only ourselves but also that mistress we call the Golden Dawn.
Why is this such a damn hard concept to understand in our post-modern era? This latest tendency amongst some outspoken authorities in the community is childish, to say the very least. It’s infantile and is filled with ugly projections. It smells awful of that old oidipean theme – that of killing your own father to acquire the Kingdom and its Queen. These individuals simply cannot accept the fact that they have to rest upon the shoulders of previous teachers’ achievements, even if they happen to be long since dead. This overly fixation on certain historical personages is an unhealthy sign, which is truly counterproductive.
Many of our previous Adepts, reformers and Chiefs weren’t that perfect and most illuminated individuals that we would hope them to be. We all know that. Probably only few numbers of them achieved that state that we long for as Adepts, to become more than human. Thus it’s easy to turn that initial idealization into literal demonisation. But as initiates we should know better than to succumb to such infantile urges; nothing necessitates this. There is no excuse for us as professed initiates to make ridicule of our ancestors, especially not in public arenas, just to attain scholarly peer recognition or to make greater sales of our books. Attain apotheosis for God’s sake (literary). Stop being scholars; start acting as true initiates and real Adepts instead.
Since writing this blog yours truly is no longer affiliated with the Hermetic Order of the Golden Dawn, Outer Order of the Rosicrucian Order of Alpha Omega® (H.O.G.D./A+O®). However, my general view on this subject stays firmly unchanged, as expressed in the above written text, and what I have authored previously on the Gyllene Gryningen blog still represents my overall opinion.

domain: literature


text:
Official announcement of The Asus ZenFone 6 has been made. We know that the previous one, the ZenFone 5 was basically just a boring iPhone X clone, but by eliminating the top bezel entirely thanks to a completely wild rotating camera, this new phone is thinking way out of the box.
At least from a design perspective, instead of chasing Apple or Samsung it seems to be determined to carve out its own niche in the phone space as the whole phone also represents a shift in direction for Asus. The whole front of the ZenFone 6 is just a seamless expanse of screen instead of an unsightly black bar or weird hole-punch dot at the top of your phone, which is pretty great to look at, although there is still a small bezel on the bottom.
Moreover, the price is the same as of less powerful ZenFone 5: the base model with 6GB of RAM and 64GB of storage starts at $499.
At first, the flipping camera may seem bizarre, especially with the wacky sound effects that Asus has set by default. But you don’t have to worry, you can easily turn it off, although the motor itself gives off its own whirring noise, but it’s actually a smart solution to the bezel issue.
A 48-megapixel main camera and a 13-megapixel wide-angle lens, these two lenses are offered by Asus, thanks to the flip system, as they can be used both for group selfies and wide-angle shots. A ton of engineering has been put by Asus into the camera. It can work as either a front-facing or rear camera as needed because it uses a customized stepper motor to rotate the lenses around 180 degrees. For improved durability, the camera housing is made out of liquid metal. However, given the number of moving parts here, there is still some concern that things could go wrong eventually. Thank fully, an automated system has been installed by Asus that will rotate the lens back if it detects that you’ve dropped the phone, but you should still probably avoid doing that.
The rotating camera also involves some clever additions. By just holding the phone and letting the camera rotate around, you can take panorama shots either vertical or horizontal. There is a motion-tracking mode for video that allows the camera to move to keep a subject in the frame.
You can also adjust the angle of the camera manually, letting you shoot with the lens at any point on the arc of rotation.
Three configurations are probably sell by Asus, of the ZenFone 6 model, starting at a 6 GB RAM/ 64 GB storage option for €499 (about $557). Two higher-specced options are also promised by the company, topping out at 8 GB of RAM and 256 GB of storage.
The specs are on par with most Android flagships, despite the mid range price, there’s a Snapdragon 855 processor, a Gorilla Glass 6 and aluminum body, a massive 5,000 mAh battery, and the aforementioned bezel-less display and flipping cameras. A 3.5 mm headphone jack is also there. Wireless charging or water resistance are the two big absences , which seem to have been cut to hit that low price point.
Asus has mercifully cut down most of its own customization, on the software side, for what it calls an “enhanced” version of stock Android, which is surely much better than Asus’ attempts over the years. A dedicated “smart key” that defaults to Google Assistant is also there, but can be remapped to activate do not disturb mode, take a screen shot, open the camera, and more.
An exact release date or price for the ZenFone 6 in the US has yet to be announced by Asus, but it should arrive sometime in the coming weeks. Between the creative design, high-end specs, and low price point, it could be a pretty interesting addition to the Android marketplace when it does.

domain: design


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text:
Coffee production for MY2019 is forecast at 3.3 million bags. MY2018 coffee production is estimated at 3.3 million bags, down two percent from the previous estimate, due to reduced moisture of the coffee bean resulting from higher temperatures at the drying patios and warehouses. Production for MY2017 closed at 3.3 million bags, down six percent from the previous estimate, due to a drought during the harvest season. Guatemalan coffee production is recovering from the rust epidemic of 2012, when 20 percent of the coffee production was lost to the disease, but the recovery and growth of the sector has been slow. In MY2017, the area planted increased after 5 years of decreases, and is revised 9 percent up to 305,000 hectares.
This is mainly due to new areas being planted with more rust tolerant plants. Coffee production represents two percent of GDP (down from a high of 5 percent), with 300,000 families depending on coffee as a livelihood (down from 600,000 in year 2000). Coffee is the major economic catalyst for economic growth in rural areas, as 20 out of the 22 departments produce coffee. Coffee rust is not the only culprit for Guatemalas lack of competitiveness in coffee: low international prices, a relatively strong quetzal compared to the dollar, and salary minimum wage that is high for the region are important variables affecting coffee production prices. In the following table, the structure of coffee costs is provided, depending on the farmers size. The small farmers represent 97 percent of the producers and 47 percent of the total coffee production. The medium farmers produce 31 percent of the total production and the big farmers produce 22 percent, representing both 3 percent of the total coffee producers in Guatemala.
Guatemala is forecast to export 3.1 million bags in MY2019, the same as previous year. Exports for MY2018 are estimated unchanged at 3.1 million bags.

domain: agronomy


** Shares of sugar companies rise 3 pct-5 pct in afternoon trade
** India, the world’s biggest consumer of sugar, scrapped the 20 pct sugar export tax to help boost overseas sales in a year of production surplus, according to a government source
** Balrampur Chini Mills Ltd rises as much as 7.4 pct, while Dalmia Bharat Sugar and Industries Ltd jumps up to 4.8 pct
** Dhampur Sugar Mills Ltd and Triveni Engineering and Industries Ltd were up more than 3 pct
** Bajaj Hindusthan Sugar Ltd and Shree Renuka Sugars Ltd were little unchanged
** Last week, Reuters reported that India would axe the sugar export tax and make it compulsory for mills to export 2 million tonnes-3 million tonnes to cut bulging stocks at home.


The epic conclusion to Chris Nolan’s Batman trilogy is an honorable ending for the trilogy, bringing this story arc of Bruce Wayne’s legacy to a close. Stepping up the scale without abandoning the outstanding performances that made this series what it is, Nolan is ambitious in his storytelling, coming full circle to meet the promises of the first film. The Dark Knight Rises implements new characters with ease and builds to a spectacular climax. Few films can so easily balance stellar performances with a grand scale, but that’s exactly what Chris Nolan has done with The Dark Knight Rises.
Taking place eight years after the conclusion of The Dark Knight, we first see Bruce Wayne (Christian Bale) as a recluse, living isolated in his spacious manor. As it was what Gotham required at the time, Batman took the fall for all of Harvey Dent’s criminal acts that take place in the last film. Reluctantly using this elegant but simple lie, Commissioner Gordon (Gary
Oldman) has helped make the city practically crime-free. Burglars and petty
thieves still exist in Gotham, but the threat of large-scale organized crime is
over. Selina Kyle (Anne Hathaway), a cat burglar and con artist, is the first
new character brought into the story. Taking on the persona of ‘Catwoman’ in the style of a classic femme-fatale, she first meets Bruce at a party in his
manor, and she continues to come in and out of his life, occasionally helping
both him and his alter ego as we build to the climax.
Christopher Nolan has outdone himself in terms of scale with The Dark Knight Rises, and while I find The Dark Knight a far superior film, there is no denying the impressive spectrum of this film. Filming primarily in New York and Pittsburgh, Nolan conducted filming scenes with thousands of extras on Wall Street, blowing up part of Heinz Field, and his team created an actual ‘Bat’ jet which they flew through city streets by hanging it from a helicopter. Nolan’s effort to do make everything in Gotham as real as possible must be admired. The co-stars are all fantastic, making a more fluid tapestry as the characters interact with chemistry. Alfred (Michael Caine) serves as the emotional heart of the film, Lucius Fox (Morgan Freeman) is the voice of reason, and Miranda Tate (Marion Cottilard) is a rich and beautiful board member who coaxes Wayne out of the shadows.
placed on the storyline. This can make the underlying themes seem
more buried then in the precursor to The Dark Knight Rises. However, the concept of pain is certainly the prevalent motif in this film. Bane tests Batman physically, causing him pain. But it’s even more about psychological pain – many of Nolan’s films deal with the human psyche. Bruce Wayne must get over his long time fears, as well as the guilt of his parents death. In order for Batman to rise above the rest, and become a symbol as he set out to be, he must abandon all fears and go through tremendous pain and sacrifice if the Batman wishes to survive.
The Dark Knight Rises has far more clichés than The Dark Knight, and the
script has some noticeable failures, yet it still feels like a succesful end to the series. Without mentioning specifics, Nolan’s ending is a little too“Hollywood” for my liking, and I feel that the last fifteen minutes of the film could be redone to be better. Other than that, there is the occasional hitch in the pacing of the film, or a cheesy line which feels very out of place in a Chris Nolan film. Yet despite these minor problems, The Dark Knight Rises is still an exceptional superhero film.
the Batman trilogy. Writer/director Christopher Nolan, along with co-writer Jonathan Nolan and cinematographer Wally Pfister, have
completed the trilogy admirably, making these Batman movies the modern generation’s answer to Star Wars. Although not as ground-breaking as Lucas’ original trilogy, Nolan’s Batman films will certainly be remembered as one of the greatest superhero trilogies of all time, and The Dark Knight will remain the greatest superhero film ever made for a long, long time. I give The Dark Knight Rises a low 5 star rating. While a great movie, it has many flaws and doesn't quite live up to the previous film.