text:
|Publication number
|Publication type
|Application number
|Publication date
|Sep 16, 2008
|Filing date
|Aug 29, 2002
|Priority date
|Nov 16, 1992
|Fee status
|Also published as
|Publication number
|Martin J. D'Souza
|Original Assignee
|The Corporation Of Mercer University
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|Patent Citations (29), Non-Patent Citations (5), Referenced by (1), Classifications (22), Legal Events (2)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation-in-part of application U.S. application Ser. No. 08/434,542, filed May 4, 1995, now U.S. Pat. No. 6,555,110 , which is a continuation-in-part of application Ser. No. 07/977,057, filed Nov. 16, 1992 (now abandoned), all of which are commonly assigned to the assignee of the present application. The disclosures of all these applications are incorporated by reference in their entirety herein.
The present invention relates to the field of drug delivery systems. Specifically, the present invention relates to methods for preparing microencapsulated drugs using non-antigenic, biodegradable materials and also to microencapsulated compositions that are targeted to phagocytic cells such as macrophages, endothelial cells, Kupffer cells, dendritic cells and the like, or a diseased organ (such as the liver, kidneys, lungs, heart, spleen), or a diseased site (such as tumors, arthritic joints), which digest the biodegradable coating, releasing the intact drug or active component either intracellularly or at the site of accumulation. Such compositions are useful in the treatment and prevention of diseases.
Microencapsulation of water-soluble compounds contained in albumin microspheres ("MS") has been demonstrated by our laboratory (and disclosed in previous co-pending applications) to target phagocytic cells such as macrophages/monocytes, which produce the majority of the pro-inflammatory cytokines. This technique has been demonstrated to improve the efficacy of cytokine inhibiting compounds such as neutralizing antibodies. We have further evaluated the method of preparation of albumin microspheres containing other categories of drugs such as CNI-1493 (a guanylhydrazone compound which inhibits p38 MAP kinase), clodronate (a bisphosphonate), antioxidants such as pyrrolidine dithiocarbamate, and antisense oligomers to NF-kB. Microencapsulation of these compounds has improved inhibition of cytokines such as TNF, and IL1-beta in an in-vitro whole blood model, endotoxin shock model, and a bacterial septic shock model. We also have evaluated the preparation and completed the efficacy testing of a melanoma vaccine preparation, which worked very well in preventing tumors in mice.
In a first embodiment of the present invention the various process parameters, materials and reaction conditions of the emulsification methodology previously developed (and described in the copending application(s) cited above) are expanded.
Drug delivery to specific diseased sites can aid in reducing side effects in patients, thereby preventing toxicity. By using drugs in a microencapsulated form, exposure of the drug to non-diseased organs and tissue can be prevented.
A methodology to produce microencapsulated monoclonal antibodies by the emulsification method, with the use of olive oil as the emulsification media, has been previously disclosed in the copending application(s). We disclose herein additional data after evaluating bioactive protein drugs, namely, anti-sense oligonucleotides to NF-kB, in several different oils as the emulsification media, and under different temperatures and we have also evaluated the process with the use of different aqueous solvents to dissolve the drugs.
In a second embodiment of the present invention microspheres are prepared by a novel nebulization method with different examples of drugs, different solvents, different temperatures and methodology variations.
Other classes of drugs evaluated using this emulsification method are as follows:
In this embodiment we have evaluated different solvents, different temperatures and methodology variations. The drug evaluated with this nebulization method is the anti-sense oligonucleotides to NF-kB.
In a particular embodiment, the present invention provides a method of encapsulating a bioactive material by nebulization, comprising:
The above delivery system shows that the anti-sense oligonucleotides to NF-kB can be used very effectively to inhibit cytokine-mediated processes involving phagocytic cells such as macrophages, white cells dendritic cells and endothelial cells. From these studies several other applications are relevant as follows:
A) Cytokine Related Diseases:
B) Vaccine Delivery System:
C) Anti-Tumor Sustained Drug Delivery System:
Sustained release of therapeutic agents for the treatment of cancer is appealing considering the fact that therapy is usually long-term. It offers the possibility of using lower doses to achieve similar therapeutic effects as conventional non-sustained dosage forms. With the advent of biotechnology and the advances in the techniques of molecular biology, our antitumor arsenal has rapidly expanded to include protein drugs, peptides and cytokines. These new weapons, although potent, still need suitable delivery systems. Being protein in nature, these agents may be targets of enzymes in the blood. As a result injecting these agents requires very high doses which are not only cost prohibitive, but also potentially dangerous. Interleukin-12 is a recently discovered heterodimeric cytokine. It has been shown in various animal models of cancers to have tremendous antitumor potential. Using genetically engineered fibroblasts, it has been demonstrated that sustained presence of lower concentrations of IL-12 produce the same antitumor effects as larger concentrations that are not sustained. However, it is not easy to produce genetically engineered cells and is even more difficult to adapt it for mass therapy in general due to the considerations of cost and the amount of labor involved. Better alternatives exist in the form of particulate drug delivery systems such as microspheres that can not only shield such protein drugs from the enzymes in the blood, but can also sustain their release. Microspheres also have the added advantage of large scale production in addition to being amenable to preparation using a wide variety of biodegradable polymers.
We have evaluated the use of biodegradable albumin microspheres to sustain the release of IL-12. When administered intraperitoneally to C57BL/6 mice bearing subcutaneous melanomas, the microspheres significantly prolonged the survival when administered at half the weekly dose of the solution formulation. The microsphere dosage form also resulted in generally lower levels of liver and kidney function enzymes, suggesting lower toxicity.
D) Transfection System:
The microspheres can be used as an effective tool for transfection of genetic material into cells. Some of the current methods of cell transfection result in a significant number of cell deaths during transfection processes such as microporation. Since the microspheres used in our studies are less than 1 micron in size, they are readily taken up into the cells and can transfer the drug/material within the microspheres directly into cells.
Other features and advantages of the present invention will become apparent upon reading the following detailed description of embodiments of the invention, when taken in conjunction with the appended claims.
The invention is illustrated in the drawings in which like reference characters or references designate the same or similar parts or parameters throughout the figures (unless otherwise noted) of which:
We will first describe the expansion of the emulsification methodology originally disclosed in prior copending application(s).
We have further expanded the testing of microspheres prepared with different drugs, oils, at different process temperatures, different aqueous solvents used to dissolve the drug and have also evaluated variations in methodology of manufacture based on the initial patent application wherein the microspheres were prepared with monoclonal antibodies to cytokine antagonists using albumin as the polymer matrix and olive oil as the emulsifying media by the emulsification methodology.
Example 1 describes encapsulation by emulsification of a representative bioactive protein, namely the anti-sense oligonucleotides to nuclear transcription factor NF-kB. Process parameter expansion included the testing of canola oil, cottonseed oil and mineral oil. The results showed that the oils tested performed well. Other bioinert vegetable and other oils, such as but not limited to, sunflower, safflower, soybean, palm, palm kernel, coconut, caster, peanut, gingley, fish, sesame, rice bran, and the like, depending on particular bioinert characteristics, and subcomponents thereof, such as, but not limited to, monounsaturated (MUFA), polyunsaturated (PUFA) and essential fatty acids (EFA), as well as mixtures of the foregoing. Other mineral oils, including, but not limited to, heavy, light and various subfractions and combinations thereof are contemplated as being within the scope of the present invention.
The temperature range of the solvent cooling was tested and broadened. Temperature range of 5-40 degrees C. was tested and found to produce acceptable results. Temperatures below about 5 degrees C. may result in at least partial freezing of aqueous components and may be undesirable.
Further, the selection of aqueous phase was expanded to now include, but not be limited to, water, phosphate buffered saline, water plus Tween� 80, and saline.
Example 2 describes the formation of microspheres of a representative tumor vaccine drug, namely, extracellular antigen, and the bioactivity obtained.
Example 3 describes the formation of microspheres of an aqueous soluble drug, namely, CNI-1493, a guanylhydrazone, and the bioactivity obtained. The test results showed that the CNI-1493 microencapsulated form using the method of the present invention was more efficacious than the corresponding doses of the soluble, non-encapsulated form in attenuating endotoxin or cytokine release.
Example 4 describes the formation of microspheres of a representative chemical drug, namely, clodronate, a bisphosphonate, and the bioactivity obtained.
Example 5 describes the formation of microspheres of a representative bioactive protein, drug, namely, anti-sense oligonucleotides to NF-kB, and the bioactivity obtained.
Example 6 describes the formation of microspheres of anti-sense oligonucleotides to NF-kB by the novel nebulizing method of the present invention, and the bioactivity obtained.
The invention will be further described in connection with the following examples, which are set forth for purposes of illustration only. Parts and percentages appearing in such examples are by weight unless otherwise stipulated. It is to be noted that unless otherwise stated the method of forming the microspheres used olive oil.
Clinical Application in Septic Shock:
Formulation and Testing of Antisense Oligomers to NF-KB
A) Introduction
NF-kB is a nuclear transcription factor, which exists in the cytosol in an inactive form complexed to IkB. Endotoxin stimulates intracellular mediators, which results in phosphorylation of IkB producing translocation of NF-kB to the nucleus with subsequent activation of DNA. The mRNA for the synthesis of multiple pro-inflammatory mediators including TNF, IL1 and IL6 is rapidly produced. We have found that microencapsulated antisense oligomers (MSASO) to the p65 subunit of NF-kB inhibits TNF, IL1 and IL6 in-vitro. Antisense compounds have the potential to be very useful therapeutic agents by virtue of their ability to inhibit specific protein synthesis. However, a limiting factor of antisense therapy has been difficulty in obtaining adequate intracellular penetration by these large compounds. Our previous work has demonstrated improved effectiveness in cytokine inhibition using antisense to NF-kB by microencapsulated intracellular delivery. Microencapsulation provides improved delivery of the antisense compound as intracellular oligonucleotides are rapidly transported to the nucleus. Our previous studies have confirmed this hypothesis by greatly improving the effectiveness of microencapsulated antisense to the p65 moiety of NF-kB in a rat model of endotoxic shock and sepsis.
B) Preparation of the Anti-Sense Oligonucleotides to NF-kB by Albumin.
1) 50 mg of human albumin was dissolved in 2 cc of one of pyrogen free water.
2) The antisense oligonucleotides (oligomers) to NF-kB was separately solubilized in phosphate buffered saline (PBS) at a concentration of 25 mg/cc.
3) The above two solutions were mixed together for approximately 30 minutes.
4) The resulting mixture was cooled to 5 degrees C.
5) 20 cc of olive oil was taken in a 50 cc beaker and cooled to 5 degrees C. and maintained at that temperature in an ice bath.
6) The mixture of albumin and oligonucleotides was added to the oil and emulsified with the aid of a Branson Sonifier at medium setting for 20 minutes.
7) The emulsion containing the microencapsulated albumin-drug microspheres were evaluated for size with the use of a laser particle sizer until the microspheres were about 1 micron in diameter.
8) The microspheres were cross-linked with 0.5 cc of a 25% w/v solution of glutaraldehyde for 1 hour with constant stirring using a tissue homogenizer at high setting while maintaining the temperature at approximately 5 degrees C. with the aid of an ice bath.
9) The microspheres were washed with three 20 cc washes of methanol and finally sized while being suspended in the final methanol wash, with the aid of sequential HPLC filters (50, 20, 10, 5, and 1 micron size).
10) The microspheres were freeze dried and stores in a refrigerator until used.
In all cases the microspheres were suspended in pyrogen free water or saline before use.
The above procedure was repeated in order to evaluate to use of different types of oils as the emulsifying media, and different temperatures on manufacture were also evaluated in addition to the 5 degree C. described above. Finally, in addition to water, different solvents were also evaluated as the media for dissolution of the drug. The specifics of each of the parameters evaluates are as follows:
a) Effect of Different Oils:
Different oils such as canola oil, cottonseed oil and mineral oil were used for the study and was compared to olive oil used previously for the manufacture of the microspheres. It is to be understood that any bioinert vegetable or mineral oil can be used.
b) Effect of Different Temperatures:
The microspheres were prepared under wide variations of temperature such as 5, 10, 30 and 40 degrees C. The method of the present invention can thus be performed at a temperature range of from about 5-40 degrees C.
c) Effect of Different Aqueous Phase used to Dissolve the Drug:
In addition to water, and phosphate buffered saline (PBS), water containing Tween™ 80 (polyoxyethylene sorbitan monooleate, available from ICI Americas, Inc.) and saline were used to examine if differences would be significant.
Drug content analysis and efficacy studies evaluating TNF-alpha suppression was conducted using the In-vitro Whole Blood Model. The following variations in the manufacture procedure were evaluated.
C) Experimental Method:
Drug content analysis was determined by HPLC methods developed in our laboratory.
The preparations were evaluated for drug efficacy with the aid of the whole blood model, briefly outlined as follows:
Blood was pooled into lavender top tubes containing EDTA. The blood was separated into three 5 ml aliquots and pre-treated for 1 hour with one of the following batches of microspheres and challenged with endotoxin (100 mcg/ml). Samples were be obtained at 0 and 4 hours post endotoxin challenge to determine the TNF-alpha levels. The efficacy of the cytokine suppression due to the addition of microencapsulated oligomers to NF-kB was compared to microspheres prepared using olive oil as described in the copending patent application.
D) Results:
Effect of Different Oils on Drug Content Analysis:
Effect of Temperature Variations on Drug Content Analysis:
Effect of Different Aqueous Phases Used on Drug Content Analysis:
Effect of Different Oils on TNF-Alpha Suppression:
Effect of Temperature Variations on TNF-Alpha Suppression:
Effect of Different Aqueous Phases Used on TNF-Alpha Suppression:
Tumor Vaccine Drug
Tumor Protection Studies Using Microparticle as Adjuvant or Coadjuvant in a Tumor Vaccine
A) Introduction
The induction of an immune response is a complex and intricate process requiring an intact immune system to evaluate. Thus, a mouse tumor model was used to evaluate the microencapsulated extracellular antigen (MECA) vaccine preparation. The antigens used in the vaccine were derived from the B16 murine melanoma cells growing in culture. The C57BL/6 mouse, syngeneic to the B16 murine melanoma cells, was used. This represents a prophylactic tumor vaccine where the mice were first vaccinated to induce an anti-tumor response. The mice were then challenged to determine if an anti-tumor response was induced with the capacity to reject the establishment of the murine melanoma.
B) Preparation of Melanoma Vaccine Preparation.
The microencapsulated vaccine preparation was made according to the method described in Example 1.
C) Experimental Methods
Immunization and Tumor Protection Studies
MECA (containing 20 μg ECA in a total of 80 μg MECA) and blank MP (microparticles) were prepared by a water-in-oil emulsion cross-linking technique using glutaraldehyde as the cross linking agent. To evaluate the anti-tumor effect of 20 μg extracellular antigen in an equivalent amount of microparticles used in the first study (80 μg MECA total), 3 groups of female C57BL/6 mice (n=5), 8-12 weeks old, were vaccinated, subcutaneously. The three groups were vaccinated with 20 μg of extra-cellular antigen (ECA) contained within a total of 80 μg of microencapsulated extracellular antigen (MECA), resuspended in a total volume of 100 μl with PBS, extra-cellular antigen in solution (ECA soln) in PBS and blank microparticles (Blank MP) in PBS, respectively. The mice were boosted every week for 3 weeks for a total of 4 injections. 7 days after the last boost the mice were challenged with 7�105 live B16 melanoma cells subcutaneously at a contralateral site, as described above. The mice were then observed for 60 days for the development of tumors and tumor size and tumor incidence was recorded.
D) Results and Discussion
Female C57BL/6 mice were vaccinated with MECA (20 μg ECA contained in 80 μg total MECA), blank MP or ECA soln subcutaneously. After the first vaccination the mice were boosted once a week for three weeks. Seven days after the last vaccination boost the C57BL/6 mice were inoculated at a distant site with 7�105 live syngeneic B16 melanoma cells. The mice were subsequently monitored for the development of tumors and tumor incidence was reported (
The studies suggest that microencapsulating tumor antigens could have an adjuvant effect in inducing tumor immunity by targeting professional antigen presenting cells. In addition, the results of the blank microparticle group of 40% tumor free at 60 days, suggests that BSA microparticles could possibly be an excellent adjuvant for the B16 melanoma due to the homology between BSA and the B700 tumor antigen.
E) Conclusion.
The in vivo dose response studies revealed that the vaccine dose of 20 μg ECA contained in 80 μg of total MECA worked very well in this study. This dose of the MECA vaccine resulted in C57BL/6 mice remaining 80% tumor free up to the 60-day study period. The studies suggest that microencapsulating tumor antigens could have an adjuvant effect in inducing tumor immunity by targeting professional antigen presenting cells. In addition, the results of the blank microparticle group of 40% tumor free at 60 days, suggests that BSA microparticles could possibly be an excellent adjuvant for the B16 melanoma due to the homology between BSA and the B700 tumor antigen.
The B16 murine melanoma tumor represents a very rigorous tumor model. For this reason it is possibly more representative of cancer in the human situations. These results do indicate that the microparticle induces a greater anti-tumor effect.
Chemical Drug, CNI-1493: a Guanylhydrazone Compound
Application in Septic Shock
Formulation and Testing of Microencapsulated CNI-1493.
Prevention of Lethality and Suppression of Pro-Inflammatory Cytokines in Experimental Septic Shock by Microencapsulated CNI-1493
A) Introduction
Endotoxemia in animals is associated with the release of pleiotropic cytokines such as TNF-alpha and IL-1-beta from the activated macrophages and polymorphonuclear cells. Experimental drugs that inhibit the effect of these cytokines such as monoclonal neutralizing antibodies (TNF-alpha monoclonal antibody), receptor antagonists (IL-1 receptor antagonist) and receptor fusion proteins have been evaluated in animals and in the clinic for their efficacy in septic shock. Recently, a newly developed water soluble tetravalent guanylhydrazone compound termed "CNI-1493" (N,N′-bis[3,5-diacetylphenyl]decanediamide amidinohydrazone tetrahydrochloride) was shown to be efficacious in reducing lipopolysaccharide (LPS) induced TNF-alpha, IL-1-beta and IL-6 release and lethality in animals.
We have previously reported studies, which demonstrated microencapsulation of cytokine neutralizing antibodies increased their efficacy compared to the soluble form in various in vitro and in vivo disease models. Similarly, microsphere form of other cytokine antagonists may also be more efficacious than the corresponding soluble form because of the targeted uptake of the microencapsulated drugs by macrophages. In this Example, we evaluated the efficacy of microsphere form of the newly developed compound CNI-1493 by Cytokine Network Incorporated. Comparison of efficacy of the soluble and microencapsulated form of CNI-1493 was evaluated using an in vitro endotoxin-induced cytokine release whole blood model, and an in vivo model of endotoxemia and E. Coli-induced peritonitis.
B) Preparation of CNI-1493 Microspheres.
The microencapsulated CNI-1493 preparation was made according to Example 1.
C) Experimental Methods
For each sample (n) blood was collected in EDTA (1.5 mg for each ml of blood) from five rats and pooled. After a baseline plasma sample the blood was aliquoted into five groups. There were six replicates in each group. Each group received one of the following treatments: saline or soluble form of CNI-1493—0.25, 0.5 or 1.0 microgram/ml or blank microspheres (MC) or MC form of CNI-1493—0.25, 0.5 or 1.0 microgram/ml. All groups were incubated at 37 degrees C. under an atmosphere containing 5% CO2. After two hours of incubation, endotoxin (100 ng/ml) 0113 obtained from Escherichia Coli (Associates of Cape Cod, Wood Hole, Mass.) was added to all groups and incubated for an additional 24 hours. Plasma samples were periodically collected at 2, 4, 6 and 24 hours after endotoxin for measurement of TNF-alpha and IL-1-beta using a modified alkaline phosphatase ELISA technique.
D) Results and Discussions
Endotoxin-induced cytokine release in whole blood model: Effect of CNI-1493 on endotoxin-induced TNF-alpha and IL-1-beta release is shown in
In vivo model of endotoxemia: The survival data is shown in Table 1.
Comparison of survival rates with soluble (Sol.) and
microencapsulated (MC) CNI-1493 in lethal endotoxemia.
Percent Survival
0 hours
48 hours
120 hours
Endotoxin alone
Endotoxin + 1 mg/kg Sol. CNI-1493
Endotoxin + ≦1 mg/kg MC form
Endotoxin + 2 mg/kg Sol. CNI-1493
Endotoxin + ≦2 mg/kg MC form
Endotoxin + 5 mg/kg Sol. CNI-1493
Endotoxin + ≦5 mg/kg MC form
Endotoxin + 10 mg/kg Sol. CNI-1493
Endotoxin + ≦10 mg/kg MC form
*Significance at p < 0.05 when compared to equivalent doses of Sol. CNI-1493 or endotoxin alone using Mann-Whitney test.
All the animals that received 1, 2, 5 mg/kg of soluble form of CNI-1493 died within 24 hours of endotoxin while 50% of the animals that received 10 mg/kg of soluble form of CNI-1493 and 25% of the animals in the group that received 1 mg/kg dose of MC form of CNI-1493 survived for seven days after endotoxin. On the other hand, all the animals (100%) in the group that received 2, 5, and 10 mg/kg of the MC form of CNI-1493 survived for seven days after endotoxin. The cytokine levels for this study are shown in
E. Coli-induced peritonitis model of septic shock: The survival data is shown in Table 2.
Comparison of survival rates with soluble (Sol.) and micro-
encapsulated (MC) CNI-1493 in E. Coli-induced peritonitis.
Percent Survival
0 hours
48 hours
168 hours
E. Coli + saline
E. Coli + blank microspheres
E. Coli + 2 mg/kg Sol
E. Coli + 2 mg/kg MC form
E. Coli + 2 mg/kg Sol.
50% (3 in 6)*
17% (1 in 6)
CNI-1493 + gentamycin
E. Coli + 2 mg/kg MC form
67% (4 in 6)*
67% (4 in 6)*
CNI-1493 + gentamycin
E. Coli + 5 mg/kg Sol.
E. Coli + 5 mg/kg MC form
17% (1 in 6)
17% (1 in 6)
E. Coli + 5 mg/kg Sol.
67% (4 in 6)*
50% (3 in 6)
CNI-1493 + gentamycin
E. Coli + 5 mg/kg MC form
83% (5 in 6)*
83% (5 in 6)*
CNI-1493 + gentamycin
*Significance at p < 0.05 when compared to E. Coli + saline group or E. Coli + blank microspheres group using Mann-Whitney test.
All the animals that received saline or blank MC or 2 mg/kg of soluble form of CNI-1493 or 5 mg/kg of soluble CNI-1493 or 2 mg/kg of MC form of CNI-1493 pretreatment died within 4 to 8 hours of E. Coli administration. There was minimal protection against lethality with a 17% survival rate after treatment with either 5 mg/kg of MC form of CNI-1493 or a 2 mg/kg of a soluble form of CNI-1493 and gentamycin. Administration of gentamycin also increased the survival rate to 50% in the group that received 5 mg/kg of soluble form of CNI-1493 pretreatment, 67% in the group that received 2 mg/kg MC form of CNI-1493 and to 83% in the group that received 5 mg/kg MC form of CNI-1493. Both soluble and MC form of CNI-1493 lowered E. Coli-induced TNF-alpha and IL-1-beta levels and the MC form of CNI-1493 was significantly better than the soluble form of CNI-1493 in attenuating E. Coli-induced TNF-alpha and IL-1-beta levels.
Microencapsulation of CNI-1493 improved effectiveness in both the in vitro and in vivo models. The results show that MC form of CNI-1493 was more efficacious than the corresponding doses of soluble form of CNI-1493 in attenuating endotoxin or E. Coli induced cytokine release and lethality. In previous studies using microencapsulated cytokine neutralizing antibodies, we saw an improvement in efficacy in inhibition of endotoxin-induced cytokine release and prevention of lethality due to endotoxin or E. Coli-induced peritonitis compared to the corresponding soluble form of the neutralizing antibodies. It may be that the effectiveness of microencapsulated compounds (excluding the sustained release form) is magnified by the intracellular release in phagocytic cells. CNI-1493 released from the microspheres after being phagocytozed by phagocytic cells provides a higher intracellular concentration that leads to effective suppression of the proinflammatory cytokines by an intracellular mechanism of action. Previous studies have shown that soluble CNI-1493 can suppress LPS induced cytokines such as TNF-alpha, IL-1-beta and IL-6 from peripheral blood monocytes as seen in this study. The mechanism by which CNI-1493 inhibits TNF-alpha synthesis is speculated to be at the translational or post-translational level. In this study MC form of CNI-1493 strongly inhibited endotoxin-induced TNF-alpha and IL-1-beta levels while the soluble form of CNI-1493 inhibited endotoxin-induced TNF-alpha and IL-1-beta levels to a smaller extent both in the in vitro and in vivo models. The extent of endotoxin-induced TNF-alpha inhibition by the lowest dose of MC form of CNI-1493 (0.25 microM) was similar to that produced by the highest dose of soluble form of CNI-1493 (1.0 microM) in the in vitro whole model. This indicates that theoretically the MC form of CNI-1493 could be at least four times as potent as the soluble form of CNI-1493 in inhibiting endotoxin-induced TNF-alpha synthesis.
MC form of CNI-1493 (2 mg/kg) provided complete protection against lethal endotoxemia while there was no survival with the same dose of soluble form of CNI-1493 (2 mg/kg) or a only 50% survival with five times higher dose of the soluble form of CNI-1493 (10 mg/kg). Complete protection by the MC form of CNI-1493 against lethality due to endotoxemia suggests a greater effectiveness of the microencapsulated delivery system. At a dose of 5 mg/kg of CNI-1493, the survival rate in E. Coli-induced peritonitis model was also much higher with the combination of gentamycin and MC form of CNI-1493 (83%) compared to the combination of gentamycin and soluble form of CNI-1493 (50%). In this infectious model of lethality both soluble and MC form did not prevent lethality except when gentamycin was used in conjunction with CNI-1493. This indicates that antibiotic treatment is essential in a severe infectious state. The experimental model of peritonitis has proven to be resistant to treatment with antibiotics alone or soluble form of TNF-alpha neutralizing antibodies alone. In fact, there has been no previously reported studies that demonstrate improved survival in this model after treatment with the soluble form of cytokine antagonists except when treated with a combination of antibiotics and microencapsulated cytokine antagonists.
In conclusion, we have demonstrated the superior effectiveness of microencapsulated CNI-1493 in suppressing endotoxin-induced TNF-alpha and IL-1-beta release using an in vitro whole blood model. This improved effectiveness has produced significantly better survival in both endotoxemia and E. Coli peritonitis model of septic shock.
Chemical Drud-Clodronate
Macrophage Depletion by Albumin Microencapsulated Clodronate: Attenuation of Cytokine Release in Macrophage Dependent Glomerulonephritis
A) Introduciton
The macrophage plays an important role in the inflammatory process through the release of cytokines, chemokines and other substances. The role of macrophage in various inflammation-mediated disease states can be evaluated by depletion of macrophages with clodronate, a water soluble compound. Clodronate, a bisphosphonate, is a potent inhibitor of osteoclast-mediated bone reabsorption and clinically used to treat metabolic bone diseases. Clodronate in free (solution) form has little effect on macrophage function following systemic administration. However, liposomes containing clodronate are readily phagocytozed by macrophages and cause depletion of macrophages in the liver, spleen, lymph nodes and peritoneal cavity, and monocytes in the systemic circulation. We have developed a method of microencapsulation of clodronate using albumin that has several advantages over the use of liposomes. Albumin can be used as the biocompatible polymer matrix to form microspheres (MS) of varying size which has greater stability and ease of preparation when compared to liposomes. Albumin is a biodegradable, non-toxic substance that has a high efficiency of encapsulation. The purpose of this investigation is to determine if albumin MS containing clodronate: 1) will produce systemic macrophage depletion, 2) have an effect on TNF-alpha and IL-1-beta release induced by endotoxin in vitro, and 3) have an effect on macrophage infiltration in experimental glomerulonephritis (GN) in rats. The results indicate that clodronate MS effectively depleted macrophages, attenuated endotoxin-induced TNF-alpha and IL-1-beta release, and blocked experimental GN induced macrophage infiltration into the glomerulus.
B) Preparation of Microspheres.
The microencapsulated clodronate was made according to Example 1.
C) Experimental Methods
D) Results and Discussion
Our study demonstrated that small doses of clodronate encapsulated in albumin are effective in depleting ED1 positive macrophages from the liver, spleen, kidney and peripheral blood in rats. Clodronate MS also produced a prompt reduction in endotoxin stimulated TNF-alpha and IL-1-beta release which was significantly greater than clodronate in free (solution) form and prevented macrophage infiltration into the glomerulus that accumulate during experimental anti-GBM GN in rat.
Macrophage depletion has been proven to be a valuable tool in evaluating the contribution of the macrophage to the development of pathological conditions. Clodronate, a bisphosphonate has little effect on the viability of the macrophage in the free form, but encapsulated into liposomes or MS (as in this study) there was a transient depletion of the macrophage population within 24-48 hours. The depletion of macrophages by clodronate liposomes was shown to be caused by apoptosis-induced cell death. We speculate a similar mechanism of action for clodronate MS.
The reduction in endotoxin-induced TNF-alpha and IL-1-beta release after pretreatment with clodronate MS as seen in this study has also been shown by others using clodronate liposomes. It has also been shown that clodronate liposomes can attenuate cytokine gene expression in mice. In the whole blood model, we also demonstrated a greater reduction of endotoxin-induced cytokine release with clodronate MS when compared to clodronate in free form. There was nearly a complete inhibition of both TNF-alpha and IL-1-beta release at the highest dose of clodronate MS that contained, not greater than 100 μg of free clodronate. The mechanism of action of clodronate MS is likely due to phagocytosis of the albumin MS containing clodronate in a similar fashion to liposomes followed by the release of the clodronate intracellularly that produces an inhibition of cytokine release due to death of macrophages. Inhibition of cytokine release by clodronate may be beneficial in the treatment of disease states characterized by proinflammatory cytokine release.
Previous studies have shown that macrophages have an important role in induction and progression of renal damage in GN. One of the hallmarks of GN is proteinuria and macrophage infiltration. The reduction in macrophage infiltration by clodronate MS in experimental GN has been previously shown by our group. We have shown that the anti-GBM induced GN causes macrophage infiltration (8.2 cells/glomerular cross-section) and treatment with clodronate MS prevented macrophage infiltration (2.2 cells/glomerular cross-section) similar to that seen in this study. In addition, we have also shown that anti-GBM GN-induced proteinuria (43 mg/24 hr) can also be significantly reduced with clodronate MS (8.4 mg/24 hr) to the same level found in normal healthy rats (5.3 mg/24 hr). Clodronate MS may be therapeutically beneficial by depleting macrophages in GN.
Effect of clodronate on endotoxin-induced TNF-alpha and IL-1-beta release is shown in
Tissue sections stained for ED1 positive macrophages demonstrate that was a significant (p<0.001) reduction of ED1 positive macrophages from liver and spleen of rats that received clodronate MS compared to healthy control rats (see Table 3).
Effect of clodronate MS on macrophage
depletion in rats with anti-GBM GN.
ED1 Positive Macrophages
Untreated Controls
Clodronate MS
280 � 23
13 � 12
38 � 2.5
13.5 � 1.5%a
1.2 � 0.1%a*
aPercentage of the total leucocytes;
*Statistical significance at p < 0.001.
There was also a significant reduction in circulating monocytes in peripheral blood (Table 3, p<0.001). Similarly, kidney sections stained for ED1 positive macrophages show that there was no macrophage infiltration into the glomerulus of normal healthy kidneys and induction of anti-GBM GN caused ED1 positive macrophage infiltration. Pretreatment with clodronate MS significantly reduced the macrophage infiltration in anti-GBM GN.
In conclusion, these studies demonstrate that albumin MS containing clodronate is an effective tool for total body depletion of macrophages in the rat. Depletion of macrophages by clodronate MS produced attenuation of pro-inflammatory cytokines and amelioration of experimental anti-glomerular basement membrane GN that has been demonstrated to be macrophage-dependent. Transient depletion of macrophages may be a treatment modality for macrophage-dependent disease state.
To each ml of blood 25, 50 and 100 μg of free clodronate in saline or 50, 100 and 200 μg of clodronate MS (equivalent to 25, 50 and 100 μg of free clodronate respectively) in saline was added. In all groups, the saline group received 50 μl of saline and blank MS group received 400 μg of blank MS for each ml of blood. Two hour later 100 ng/ml of endotoxin was added and the blood was incubated for 24 hours in an atmosphere of 5% CO2 at 37 degrees C. Plasma levels after endotoxin challenge is shown in this figure. The MS form of CLON attenuated endotoxin-induced TNF-alpha levels significantly better than the free form of CLON at p<0.05 level.
Bioactive Protein Drug NF-KB
Application-Septic Shock
Method of Preparation-Emulsification Method
Preparation of Microspheres Containing Cytokine Antagonist Namely Anti-Sense Oligomers to NF-KB (Bio-Active Protein Drug)
Evaluation in Whole Blood Model, Endotoxic Shock Model and Peritonitis Model
Microencapsulated Antisense Oligomers to NF-Kb; a New Approach to Pro-Inflammatory Cytokine Inhibition
A) Introduction
Inhibition of individual protein synthesis is possible by antisense oligonucleotides after binding with its specific mRNA. However, inadequate intracellular penetration of antisense compounds has limited their effectiveness. Antisense compounds contained within microencapsulated albumin takes advantage of the normal phagocytic function of macrophages to deliver antisense oligonucleotides intracellularly for improved exposure of the oligomers to nuclear and cytosolic mRNA. Flourescein labeled oligonucleotides when microinjected into macrophages appears in the nucleus within minutes, thus interacting immediately with synthesized mRNA.
Studies done in our laboratory have demonstrated that albumin microcapsules 1) are rapidly phagocytozed by macrophages in-vitro and in-vivo 2) are distributed to over 90% of monocytes/macrophages in the liver, spleen, kidney and blood and 3) migrate to the area of infection. In previous studies, we have demonstrated improved efficacy of micro encapsulated neutralizing antibodies to TNF and IL1 in both in-vitro cytokine suppression and animal survival using an in-vivo fatal endotoxic shock model and a peritonitis model of infection. Thus, microencapsulated drug delivery directly targeting the macrophage, which secretes the majority of proinflammatory cytokines, may improve the efficiency of these compounds.
NF-kB has recently been described and is thought to be the nuclear transcription factor responsible for the synthesis of proinflammatory cytokines such as TNF and IL1. Other substances involved in the inflammatory process are also regulated by NF-kB. Increased activity of NF-kB has been described in sepsis and in other inflammatory conditions such as glomerulonephritis, acute respiratory distress syndrome, and inflammatory bowel disease. Thus, antisense oligonucleotides to NF-kB may alter the inflammatory response by suppressing the synthesis of the proinflammatory cytokines. Microencapsulation of these compounds may further improve efficiency by direct macrophage targeting.
The aims of the present study are as follows:
a) to determine if albumin microencapsulation of antisense oligomers to NF-kB will improve suppression of TNF, IL1, IL6 and IL8 to endotoxin stimulation in an in-vitro whole blood model and
b) to determine if microencapsulated oligomers to NF-kB will suppress proinflammatory cytokines and improve survival using in-vivo endotoxic shock and peritonitis models.
B) Preparation of Microspheres.
The microencapsulated anti-sense oligonucleotides to NF-kB were made according to Example 1.
C) Experimental Methods
Samples of blood were drawn from normal human volunteers and separated into multiple 1 ml aliquots. 100 ug of E. Coli endotoxin was added to each specimen. Cytokine levels were determined by ELISA in duplicate in each group after the following incubation times: TNF—4 hours, IL1—24 hrs.
The following groups were studied:
1. control: endotoxin+saline
2. NF-kB antisense in solution, 200 and 300 ug/ml given 1 hr prior to the addition of endotoxin
3. NF-kB non-sense (scrambled) 200 and 300 ug/ml
4. Microencapsulated antisense oligomers to NF-kB 200 and 300 ug/ml
5. Microencapsulated non-sense (scrambled) oligomers 200 and 300 ug/ml
Endotoxic shock was produced in Fischer rats weighing approximately 150 grams by intravenous injection of 15 mg/kg of E. Coli endotoxin. TNF was measured by ELISA at 0, 4 hrs, 8 hrs, 24 hrs and 48 hrs. Survival was observed for 5 days (120 hrs).
After a dose response study was performed, 300 ug of microencapsulated antisense oligomers to NF-kB was injected into 10 rats and 300 ug of oligomer in solution were given intravenously to 10 rats.
Peritonitis was induced in rats by the intraperitoneal injection of 1010 organisms of E. Coli. gentamycin 15 mg/kg was given intraperitoneally for 3 consecutive days. TNF was measured at 0 hrs, 4 hrs, 8 hrs, 24 hrs, and 48 hrs. Survival was observed for 5 days (120 hrs).
1. Simultaneous treatment: E. Coli peritoneal injection and the following treatments were given simultaneously and then daily for an additional 2 days.
a. control
b. microencapsulated NF-kB I.V. 400 ug/rat n=10
c. microencapsulated NF-kB I.V. 200 ug/rat n=10
d. solution NF-kB I.V. 400 ug/rat n=10
e. solution NF-kB I.V. 200 ug/rat n=10
2. Delayed treatment: Treatment with the above doses of oligomer initially given 4 hrs after the dose of intra peritoneal E. Coli (at the peak TNF level) and then for an additional 2 days.
D) Results and Discussions:
Microencapsulation of antisense oligomers to NF-kB improves pro-inflammatory cytokine inhibition by increased intracellular penetration into the macrophage. Microencapsulated antisense oligomers to NF-kB inhibit TNF>IL1>IL6>IL8 to a greater extent than equivalent amounts of oligomer in solution (p<0.05) using the in-vitro whole blood model. Microencapsulated NF-kB oligomers produced a dose dependent improvement in TNF inhibition in the endotoxic shock model in rats. 80% survival at a dose of 300 ug per rat was observed compared to 20% in the endotoxic shock model with an equivalent dose in solution (p<0.05). Microencapsulated oligomers produced 80% survival in the peritonitis model and 70% survival in the delayed treatment group compared to 30 and 20% in the solution group respectively. TNF and IL1 were inhibited to a greater extent in the microencapsulated group. Survival occurred in the delayed treatment group with microencapsulated oligomers even when given after the peak in TNF occurred.
In summary, microencapsulated oligomers to NF-kB improve pro-inflammatory cytokine inhibition both in-vitro and in-vivo with improved mortality in otherwise fatal models of endotoxic shock and peritonitis. Microencapsulated oligomers to NF-kB may be of value in the treatment of pathological conditions characterized by pro-inflammatory cytokine activation.
E) Results:
A) Introduction:
We were interested in evaluating microspheres prepared by a nebulization method of different categories of drug types (bio-active proteins, oligonucleotides, chemicals, vaccines) and also to study the effect of variations in oils, temperature and methodologies on the preparation of microspheres. We used a biodegradable non-antigenic albumin matrix to microencapsulate the cytokine antagonists [such as anti-sense oligomers to NF-kB].
B) Experimental Methodology
Microsphere Preparation:
Nebulization Method:
General Methodology:
The general basis of this invention involves the following steps:
An aqueous solution of the drug (which may be a bioactive protein, drug or a synthetic drug) to be microencapsulated is prepared along with the encapsulating polymer (such as, but not limited to, albumin, chitosan, globulin or some other bio-degradable natural or synthetic polymer). The polymer-drug solution is then aerosolized (with the aid of some spray forming device, such as, but not limited to, an ultrasonic nebulizer) to form a fine mist-like spray. This mist or spray containing the polymer-drug (or other material) solution is then directed into a solvent system such as butanol or other lower carbon alcohol, such as, but not limited to methanol, ethanol, propanol, and the like (see
Some of the advantages of this method are as follows:
Bioactive Protein Drug NF-KB
Application-Septic Shock
Method of Preparation-Nebulization
Preparation of Microspheres Containing Cytokine Antagonist Namely Anti-Sense Oligomers to NF-KB (Bio-Active Protein Drug) by the Nebulization Method
A) Introduction.
Microspheres containing the cytokine antagonist [(anti-sense oligomer to NF-kB (bio-active protein drug) were evaluated in this study.
B) Preparation of the anti-sense oligonucleotides to NF-kB by albumin using the nebulization method.
Microspheres containing the cytokine antagonist [(anti-sense oligomer to NF-kB (bio-active protein drug)] were cross-linked to the albumin microsphere matrix.
In all cases the microspheres were suspended in pyrogen free water or saline before use. In the nebulization step, the particles created at the spray head were conducted via a tube to the container containing the solvent in step 5) above and the tube tip was maintained below the surface of the solvent so that the nebulized particles were introduced into the solvent solution below the air interface surface so as to minimize loss to the atmosphere.
The above procedure was repeated in order to evaluate to use of different types of oils and solvent systems as the emulsifying media, and different temperatures on manufacture were also evaluated in addition to the 5 degree C. described above. Finally, in addition to water, different solvents were also evaluated as the media for dissolution of the drug. The following variations were evaluated:
Different oils/solvents such as olive oil, cottonseed oil, canola oil, mineral oil and butanol were used for the study.
The microspheres were prepared under wide variations of temperature conditions.
In addition to PBS, saline, distilled/de-ionized water and water with Tween� 80 were used to dissolve the albumin and the drug.
The effect of cross-linking was evaluated where the cross-linking agent is added after all the microspheres are atomized into the solvent.
C) Experimental Methodology:
Drug content analysis was determined by HPLC methods developed in our laboratory.
The preparations were evaluated for drug efficacy with the aid of the whole blood model, briefly outlined as follows: Blood was pooled into lavender top tubes containing EDTA. The blood was separated into three 5 ml aliquots and pre-treated for 1 hour with one of the following batches of microspheres and challenged with endotoxin (100 mcg/ml). Samples were obtained at 0 and 4 hours post endotoxin challenge to determine the TNF-alpha levels.
D) Results:
Chemical Drug Pyrrolidine Dithiocarbamate
Application-Septic Shock
Method of Preparation-Emulsification
Preparation and Evaluation of Microencapsulated
Pyrrolidine Dithiocarbamate
A) Introduction: Pyrrolidine dithiocarbamate (PTDC) is a water soluble, low molecular weight antioxidant substance, which inhibits NF-kB activation. NF-kB is the nuclear transcription factor, which is responsible for the activation of pro-inflammatory cytokines. Several studies have demonstrated the effectiveness of PTDC in cytokine inhibition in-vitro as well as improving mortality in endotoxic shock models in rats. We have demonstrated the improvement of the efficiency of compounds such as neutralizing antibodies and antisense oligomers to NF-kB in cytokine inhibition both in-vitro and in-vivo. Microencapsulation of a compound targets the macrophage and improves the efficiency of cytokine inhibition.
The whole blood model will be used to evaluate the efficacy of the microencapsulated PDTC. Three doses [15 micromoles (uM), 30 uM, and 60 uM] will be studies studied. These doses will be added to 1 ml aliquots in both encapsulated and solution form. TNF alpha will be measured by the standard ELISA procedure.
Although only a few exemplary embodiments of this invention have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims. It should further be noted that any patents, applications and publications referred to herein are incorporated by reference in their entirety.
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|Fee payment
Year of fee payment: 4
|Oct 16, 2015
|Fee payment
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Mumsnet has not checked the qualifications of anyone posting here. If you need help urgently, please see our domestic violence webguide and/or relationships webguide, which can point you to expert advice and support.
NC with PIL for 10 years and now we've had a text...(109 Posts)
We were married for 10 years before DH decided enough was enough and were always walking on eggshells. It was so easy to cause offence. e.g. MIL called when DS1 was 3 days old to complain that the Mother's Day card we sent was too small and there were loads of similar things.
What usually happened was that they would go off in a sulk and after a few days I or DH would call, smooth things over and we'd be back to normal, until the next time.
10 years ago, after MIL was "devastated" because my mum organised something lovely for DH's birthday (mum really loves her SIL ) DH decided he'd had enough and we didn't make the usual call. Although there have been occasional (often not very nice) emails since he hasn't seen or spoken to them in 10 years. They haven't seen their GC since they were 2yo and 6mo.
Anyway this week he's had a text. Their Golden anniversary is coming up. Will we go, let them know so they can make the booking? No mention of what the celebration is. No idea if it's just us, immediate family or a huge celebration.
I've said to DH I'll do whatever he wants but that I don't think meeting up for the first time in 10 years, in front of loads of people who (presumably) know we haven't spoken for 10 years is a good idea. If he/they want to reconcile there must be better ways...
He has decided he doesn't want to go and doesn't want to see/speak to them so he's going to decline by text.
He wants to send a lengthy text about why it's a bad idea. I think it would be better just to say no thanks.
MIL will say "I love you" and then call to berate you because you don't love her enough because of some small slight.
This is pathetic. The whole thing of being jealous of her own children because they had things she didn't have is equally pathetic and whatever anyone's justification for bringing children into the world is, it should never be to compete with them, force your bitterness on to them for the way your life turned out, expect them to 'pay you back' for bringing them up etc...
OP and her DH are absolutely right to stay NC. Are people just glossing over the fact that because OP and her DH didn't ring these people over their latest unreasonable hissy fit, they didn't make contact for ten years? And let's make no mistake about it, it was they who made no contact. It was they who couldn't look at their action and say 'ok were we being a bit petty there? Are we not big enough to extend an olive branch'?
If people want to stay in contact with toxic immature people who place unreasonable expectations on them and did an overall crap job of parenting, that's a personal choice. But it's not for everyone and that choice can't be projected on everyone else.
OP see their action for what it is: an attempt to save face at a family event where they'll undoubtedly have to explain their ten years' radio silence (and that explanation will make them look like arses to any listener if they tell it truthfully).
Only if they come to you with an abject apology and a commitment to change, separately to any event that happens to be coming up, should you consider engaging with them... and even then, only one chance.
"Ok, we get the message, life's too short to bear grudges and everyone would love to see you but we won't bother you again"
i agree - there is nothing NICE about this message:
Ok, we get the message, = agressive
life's too short to bear grudges = "life's too short for YOU to bear grudges/Get over it already
everyone would love to see you = We need you to make us look good
we won't bother you again" - a pathetic attempt to guilt trip or scare DH into buckling.
Oh yes, not a single word of that message was in any way shape or form an olive branch.
If they had have written, 'Can we talk about this?' it would have been a whole different matter.
There is no contrition, there is no climbing down, only guilt, threats and aggression.
^^^^What Hissy said spot on.
Where's the effort in of couple of texts. Why not go round and ask if they can have a talk, see if anything could be done to resolve things. We love you and miss you and we are sorry its taken so long....
why has it taken them so long? Id be begging my ds if he refused to talk to me.
Life is too short to bear grudges. So ... er ... where were they the last ten years?
Meerka- playing the victims of course!
... and bearing the grudge <dryly> oh boy, some people should have mirrored lenses on the inside of their spectacles shouldn't they. Assuming they wear specs.
Message withdrawn at poster's request.
Op, it sounds as though you both did the right thing by going no further with this.
The key is in the message "tell EVERYONE......" Well it sums up exactly why they wanted to see you, so they could show EVERYONE else and parade you all!
There are personality disorders which stem from genes rather than upbringing; mental health problems too. More and more is being discovered about how the brain works. I doubt very much that unconditional love from parents, or anyone else, would be able to counter something that due to genetics.
There are environmental factors too of course. An acrimonious divorce can result in one parent using the children to get at the other parent, which can result in parental alienation - and not always towards the NRP; sometimes it's the NRP who manages to alienate the children towards to the RP. Sometimes children reject the RP because the RP has been forced to allow contact against the wishes of the children, which the children cannot or do not forgive. There are almost as many possibilities as there are families.
If a child is born with a predisposition of some sort, which is then fanned and enabled by environmental factors which may be beyond the parents' control, then that can hardly be blamed on the parents; and yet that child may well grow up to thoroughly toxic.
There is research which shows that parental influence wanes tremendously as the child grows. Can you blame a parent when the things which are hurting their child are completely unknown to them, and the people who should know - a school for instance - do not know either? How many of us blamed the parents for what happened in that nursery a few years ago, where one of the employers was a paedophile? And yet, how do we know how those babies might be affected long term by that experience?
It is simplistic to say that children can only become toxic towards their own parent(s) because the parent(s) were lacking in some way. We're none of us perfect, after all.
OP, I think Springy was just trying to put the other side of the argument. I don't think she was criticising your or your dh particularly, just wanted to help you see all aspects of your options, before you made the final decision - and after all, with an important decision you do want to as much as you can about the subject, don't you? Most of us want to find out everything we can about something and listen to all povs before making a big decision.
Mind you, I see that it is no longer relevant anyway. And your dh was never keen on seeing them again either.
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text:
Vina Yokone
(Japanese: Yokoneビニャ- Yokone Vina)
Last name in japanese: (Yokone: Rough translation for first year)
First name in japanese: (ヴィーナ: Rough translation for stringed instrument)
Type: UTAUloid
Model: Forgotoloid
GENDER Female VOICE RANGE N/A RELATED CHARACTERS
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Saigai Haisha (Friend)
Nippoid (love interest)
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WEIGHT 150 lbs. CHARACTER ITEM Spirits CREATOR YokoneVina
HEIGHT 6' 4' VOICE SOURCE YokoneVina PICTURE LINK DeviantART
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Additional Info:
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Eye color: emerald
Outfit colors: Black/purple/gray/white
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NOTE: The information contained herein is provided as general information only and has been obtained from sources deemed reliable. It is provided without any guaranty, warranty or representation, expressed or implied, made by Lands of America, or any related entity, as to the accuracy or completeness of the information. The information is presented subject to errors, omissions, change of price or conditions, prior sale or withdrawal without notice. Prospective purchasers should make their own investigations, projections and conclusions concerning the information.


text:
Makila, P.M. and Partington, J.R. (1992) Robust identification of strongly stabilizable systems. IEEE Transactions on Automatic Control, 37 (11). pp. 1709-1716. ISSN 0018-9286
For strongly stabilizable systems for which a strongly stabilizing controller is known approximately, the authors consider system identification in the graph, gap, and chordal metrics using robust H ∞ identification of the closed-loop transfer function in the framework proposed by A.J. Helmicki et al. (1990). Error bounds are derived showing that robust convergence is guaranteed and that the identification can be satisfactorily combined with a model reduction step. Two notions of robust identification of stable systems are compared, and an alternative robust identification technique based on smoothing, which can be used to yield polynomial models directly, is developed
|Copyright, Publisher and Additional Information:
|Copyright © 1992 IEEE. Personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution to servers or lists, or to reuse any copyrighted component of this work in other works must be obtained from the IEEE.
|The University of Leeds
|Academic Units:
|The University of Leeds > Faculty of Maths and Physical Sciences (Leeds) > School of Mathematics (Leeds)
|Depositing User:
|Sherpa Assistant
|Date Deposited:
|30 Sep 2005
|Last Modified:
|04 Jun 2014 17:57
|Published Version:
|Identification Number:


text:
Jonathan Charles The Aberdeen Stool
$ 759.00
Without question it's essential to be able to pull up extra seating at a party. I find my house dotted with Aberdeen and her sisters. I use them as an occasional cocktail table or a small place to put my "book on the go".
- Collection: William Yeoward Collected - Country House Chic
- Finish code: FIN-OAK-10-1 (Greyed Oak)
- Dimensions: 13" w x 13" d x 17" h
- Weight: 13.23 pounds


text:
It was at this moment that "the big fade" occurred for Army's overall fortunes.
Williams threw a fade pass to the left side of the end zone. The fade is a risky play in the sense that it relies on the receiver to out-jump the defender, but for a growing and learning quarterback such as Williams, it's a safe throw because it's supposed to involve a simple decision-making process. You see a receiver in isolation against a cornerback, and you throw the ball in the air. Not too complicated. When pressed for a big play inside the red zone, the fade route is a fairly common play called by offensive coordinators around the country. One can't fault the play selection from Army's offensive braintrust.
There was just one problem with this play, however: Williams--perhaps too intent on wanting to throw the fade--lofted his pass into double coverage. Given that the side of the end zone acted as something of a third defender, one could say that Army's quarterback challenged triple coverage on the play. This is not the kind of scenario in which you throw a fade. With single coverage, the fade is an excellent read and a smart decision. With double coverage from the defense, the fade is a terrible read and a poor decision.
Williams' fade pass was, of course, intercepted, and after two and a half additional quarters of football, it became painfully apparent that the Black Knights' last, best and only chance to take the lead over Georgia Tech came on that ill-fated fade. Army got the play call it needed, but the good read was lacking. In money situations, you need all facets of an equation to work in your favor; getting merely half of the formula right isn't good enough.
Perhaps the outcome of this game would have been different if the big play of the second half--a controversial Georgia Tech interception that very possibly could have been an Army reception--had been reviewed and then overturned by replay. (Tech's Jahi Word-Daniels seemed to wrestle the ball away from a Black Knight receiver only after hitting the ground. The principle of "simultaneous possession going to the receiver" could have been invoked, thereby enabling Army to keep possession of the ball. The play might have been an interception, but the bottom line is that the episode demanded more replay scrutiny than it ultimately received.) But that controversy aside, the fact still remains that Army had Tech on the ropes in the second quarter, only to allow the Yellow Jackets to rise from the canvas. Once the breaks went against Army in the second half, the Black Knights didn't have any margin for error. Despite playing an opponent whose offense had lost its meal-ticket workhorse, the boys from West Point couldn't take advantage.
Carson Williams and the passing game are better than they were in September, but the fact remains that Stan Brock's air attack is still not making game-changing plays in crucial situations. Kevin Dunn tied the Tulane game with a Hail Mary, but football teams won't compile winning seasons on a wing and a prayer. You have to execute basic plays and make sound reads in major moments. Third and goal from the 8 in a tight game qualifies as a major moment.
Army is still battling and scrapping. This team isn't about to fade away anytime soon. But this team's fortunes won't improve until fade routes--and other plays, of course--are carried out with greater precision at crunch time.


text:
Al Meyerhoff, formerly of the Natural Resources Defense Council and now a trial attorney at the notorious firm of
Lerach Coughlin Stoia, makes the case for Democratic obstruction of Bush's remaining appellate nominees in the Legal Times. There's not much to Meyerhoff's argument beyond "they did it too," and complaints that a majority of sitting federal judges were nominated by Republicans. (Well, what do you expect when there have been Republican Presidents making nominations for 20 of the last 32 years?)


text:
Glasgow goes quiet
Dumfries and Galloway members led the unsuccessful attempt to commit the union to a ballot in October. John Dennis, a local activist, said: "Talk will get us nowhere."
Willie Hart, the Glasgow local secretary and chair of the union's finance and general purposes committee, ridiculed the idea of moving against a Government that had only been in power for four weeks.
But Joe Eyre, Glasgow and past president of the College Lecturers' Association, said it was "a bit tiresome and disingenuous" to be constantly told the time for action was never appropriate.
Mr Eyre distanced himself from Mr Dennis's remarks. "We want to talk but, when we talk, we want people to take due account of what we say."


text:
Iam still in consultation stage ...outpatient surgery in clinic was such frightening thought to me until i met an american board certified plastic surgeon and his fees as outpatient are the same as fees as in patient for non ABPS , but really u can notice difference he gained my trust instantously and poured me with advices to reach perfect shape and advised me to do them when i reach my perfect weight and was 100 percent sure of results he can create and proud of all his work ! What do u think
Outpatient Surgery Done by ABSP Vs Inpatient Done by Non ABPS?
Doctor Answers 2
Choosing the right surgeon
It is your choice, and I think you already made it. However, I will caution you that nothing is a 100% certain guarantee.
Board Certified Plastic Surgeons are the MOST important factor
It doesn't really matter where the surgery is done as long as it is a certified facility. The MOST important factor is going to Board Certified Plastic Surgeon. It is important to see if he is board certified by the American Board of Plastic Surgery. You want to make sure that the surgeon you chose has adequate experience with that procedure and good results by talking to former patients.
These answers are for educational purposes and should not be relied upon as a substitute for medical advice you may receive from your physician. If you have a medical emergency, please call 911. These answers do not constitute or initiate a patient/doctor relationship.


text:
Space technology
Print advertisment created by Sir J.J. Institute of Applied Art, India for National Geographic, within the category: Media.
Art Director / Copywriter / Illustrator: Abhijit Kalan
mejor veo discovery channel Zzzz
I really am not feeling the National Geographic brand here. Boring art direction. So-so copy. Feels like a first idea. Just keep going. There's an idea here, but you can run with it or not. I, personally, wouldn't have played with it.
You have to hate advertising in order to truly love it.
this execution is more relevant. but i don't think headline is needed.
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text:
eyes are open
-anything visual
-nerd shit
i love its always sunny in philadelphia, game of thrones, GIRLS is a show i love to hate- but i keep watching like the challenger exploding, Anything with zombies is perfect.
Al Green, Kanye, honeyhoney, Jack White, Justice, Slick Rick- those were the last songs i played today. Kendrick Lamar is nice.
I love Pizza but most of it sucks here :(
the internets
sleeping in
a phillies hat
a car with air conditioning
iced americanos
and if you're not like this girl,


text:
Yo All, MDM here ..
Ok I just read the new One Piece chapter 666 and if you have yet to read it then please drag your lazy("sexy" in-case of ladies) a$$ to nearest manga-website and read it >_> don't blame me for spoilers later (_ _ ) .. or I will FLIP you instead >_>
http://i1189.photobucket.com/albums/z438/turtlecorn/tumblr_lu6cpy9pA11r1cnsr.gif http://i1207.photobucket.com/albums/bb479/USS-Special/shock/OMG%20FREAKING%20OUT%20AND%20TABLE%20FLIPPING/tumblr_lnzqr1TM6S1qaovwd.gif
Yes, .. lots of TABLE-FLIPPING and lots of WTFs are happening in this chapter >_> .. I don't even know where to start >_> .. here goes for the title of KING OF TABLE FLIPPING..
- WTF, Why are Zoro, Sanji and Brook sleeping like babies after doing boo-boo =_= don't tell me I waited for 2 weeks to read this chapter only to know that you got your ass kicked my some Hip-of-a-fail brother pair >_> shame on you 3, go DIE!! .. oh, you already did (_ _)
- WTF, why is Monet sitting amon great men?? GO IN THE KITCHEN AND MAKE A SANDWICH DAMMIT!!
- WTF, WHATS WRONG WITH MONET's GLASSES?? Monet, are you trying to fit-in? or wanna look "cool"? I mean whats up with the "hypnotise" shades >_>
- WTF, WHY MONET IS ALWAYS READING AND WRITING?? hey, are you a book-worm-nerd?? Speak now or forever hold your peace women cause I have some high-expectations for you >_>
- WTF, pointing gun at Law? .. really Ceaser? I mean, you better shoot something that hurts of you will end up getting sold on pork-meat-shop diced.
- WTF, LAW came to Punk Hazard alone? =_= where is you crew dammit? you better not done something bad to them to become Shichibukai .. .. especially Bepo, I am rooting for him to become the next Pirate King (*^*)
- WTF, when did Joker left Gotham City and came to One Piece side? Why am I the only one who didn't know about this? WHYYYYYYYYYY
- WTFFFFFFFFF, Monet .. you had such fine pair of sexy legs and you let that experiment-fail Clown to fix those chicken legs on you? I mean WTF??? You had a good chance to rank somewhere around 5th to 8th in my Favorite One Piece Women right-after kalifa but no more .. .. you can't walk in my heart with those legs >_> .. I am vegetarian (_ _ )
- WTF, SH's temporary base got attacked? ODA!! MAKE UP YOUR MIND DAMMIT!! This arc is getting really randomly-complicated >_>
- WTF, NAMI DON"T MOCK BEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEM, BEEM is the ancient weapons Uranus .. .. just saying.
- WTF, 4 pages worth of BULLSHIT!!!!!! (skips its)
- WTF, Luffy got there and they got away? WHERE IS LOGIC?? Luffy, where is your haki? What the hell Rayliegh thought you?
(reads the last section where Law walks around like a BOSS)
Ok drama-queening aside, this was total random and WTF chapter and I hope the upcoming chapters are little more One Piece like >_> If you share the same opinion as me on this chapter being a WTF-friendly then please flip a table(copy it from title and paste it on your comment)
This WTF blog is over and this MDM whose really sick with terrible cold is out.


text: This is being discussed in the Cleaning-Up Spam thread so I figured I would make this a proper suggestion. If XenForo uses a collaborative, centralised spam database where we can submit users of our forum for analysis (IP check of a blacklist, e-mail check) and report a user (submit their IP and e-mail for analysis, if it meets a criteria add them to blacklist) we could create a very efficient spam database that is tweaked and suited to everyone from XenForo. This database would make people feel like they are participating in XenForo and it can work in conjunction with the Spam Cleanup function that is already built in. Another option could be to have automated spam checks on the forums (which you can opt out of) which basically just scans the forum for any suspicious spam-like activity (such as having links in profile fields, having >X links in a post, etc). Discuss P.S: Like the post if you agree - new prioritising method


text:
I can't find them anywhere for a decent price....only thing I'm finding is at Point Loma Seafoods and they are going for $6.50+ per. My parents were getting them back home (mid-atlantic region, granted) for $2 a pop for Whales. Got me thinking how badly I need to find them here in SD. Anyone have any clue?
Point Loma Seafoods
2805 Emerson St, San Diego, CA 92106


text:
- "There's a big race tomorrow, on Boonta Eve."
- ―Anakin Skywalker, to Qui-Gon Jinn
The Boonta Eve was an annual holiday and festival celebrated on the desert planet of Tatooine during the last decades of the Galactic Republic. It honored the rise of Boonta Hestilic Shad'ruu to godhood.[1] The Hutts, who dominated Tatooine at the time, would host the Boonta Eve Classic Podrace to commemorate the Boonta Eve.
- Star Wars: Episode I The Phantom Menace (First appearance)
- Star Wars Journeys: The Phantom Menace
- Star Wars: Commander
- Boonta Eve Classic Podracer Pilots in the Encyclopedia (link now obsolete; backup link on Archive.org)
- Boonta Eve Classic Podracer Pilots in the Databank


text:
Is Digital Marketing destroying your business?
Learn a thing or two about innovation from school students! Marketing Guru Gary Hamel shares his eye-opening experience of working with a group of high school students as a part of program, dubbed INTRSCT, designed to teach innovation skills. Read this interesting antithesis by Neil Clair


text:
Dr. Bradley Barnett MD Patient Reviews
- Family Medicine Tucson, AZ
Overall Rating
37 Patient Reviews
- Friendly Staff
- Promptness
- Wait Time 32.4 minutes
- Appropriate Followup
- Spends Time With Patients
- Bedside Manner
- Fair and Accurate Diagnosis
- Easy Appointments
Showing 1 - 12 reviews
5.0 of 5
December 15th, 2015
Knowledgeable and caring provider. Both my wife and I go to him, and he takes the time to discuss everything. Personable and very professional.
5.0 of 5
April 5th, 2015
The finest most friendly physician I have ever encountered - his "medicine" is of equal value.
2.3 of 5
January 22nd, 2015
Leaving after 15 years
I have been Dr. Barnett's patient for 15 years, and can agree whole heartedly with the other reviewers that he is a very compassionate and knowledgeable doctor. However, the atmosphere and overall care at his office has declined significantly over the last several years. Most of your visits will begin with the rude front office staff, followed by a rushed visit with his PA. It is impossible to get an appointment with Dr. Barnett, unless your ailment can wait a couple of months. I am afraid that dealing with his incompetent staff is no longer worth the return.
2.3 of 5
July 10th, 2014
no change
i was hoping that just maybe he had hired a new staff. he is a wonderful doctor but you better have alot of time i never got out in less than 1 hour
5.0 of 5
December 13th, 2013
Great Doctor - not so great office
Wonderful physician I have been a patient of Dr. Barnett's for years. He was my mother's Doctor also and we couldn't have had a better doctor for my mother Who had Alzheimer's for 10 years. He is the most caring and compassionate doctor I have ever come in contact with. Now for the unfortunate part, The office staff, not very courteous. When trying to reach the office manager, good luck. They will not transfer you unless you tell them your specific reason for calling. If you tell them it's of a personal nature, they will not transfer you. I do not want to discuss anything personal with the receptionist answering the telephones. To make matters worse, there is no way of contacting Dr. Barnett, they run a tight interference that makes it impossible. Bottom line: Great doctor. Poor staff. Poor office functionality. It's a shame that after all these years I feel I must look for another doctor. Sadly, Dr. Barnett will never know why, because I'll never be able to reach him. I guess I could pay out-of-pocket to Schedule an appointment to talk to him about these matters.Normally, I would say that's not worth it, however, Dr Barnett actually is worth it.
5.0 of 5
August 19th, 2013
A Very Happy Patient
Dr Barnett is the Best of The Best--He listens to you-He cares about you- He takes time for you-He never makes you feel that he has another patient waiting-He is wonderful
1.0 of 5
February 21st, 2013
Doctor Barnett is wonderful. It is front office and billing personnel That leaves a lot to be desired. The front staff does not give off a friendly and open vibe. This office does not send out billing statements but expects payment in a timely manner? I have requestedbilling statements. They just won't do it. Confusing to me?
2.3 of 5
February 14th, 2013
Doctor fine, Staff rude and disorganized
I am so frustrated with this doctor and mostly with his staff. I am leaving and going to another highly recommended Family Practicioner, suggest you do the same. I have repeatedly asked to have my prescriptions renewed, left messages and never received call back. I am done!
1.0 of 5
April 10th, 2011
I recently injured my knee while hiking. I was completely unable to walk or drive due to the pain. Even though I have been a patient of Dr. Barnett's for years, I was told the earliest I could get an appointment was 5 weeks away. I tried to explain how much pain I was in, I was told that they would leave a message for Dr. Barnett and he would decide if he wanted to see me or not. I found another doctor and am very happy with the change!
5.0 of 5
December 14th, 2010
I found Dr Barnett to be especially caring. He's quite competent. My favorite GP that I've been to (and I've been to many over the years, having moved a lot).
3.7 of 5
June 3rd, 2010
Dr Barnett is an excellent doctor. However, he takes on too many patient so that he is always busy. He needs a good staff to support him which does not exist right now.
1.0 of 5
January 6th, 2010
I should have believed the ratings for this doctor. Staff was unfriendly and didn't seem to care. I waited an hour and a half before walking out. No one had the common courtesy to tell me the doctor was tied up with another patient. When I walked out the staff had no response, no one asked me why I was leaving. I'm sure Doctor Barnett is a good doctor and well qualified but he needs a new staff!
Health Guides
Type 2 Diabetes
Type 2 diabetes is a chronic disease in which your body is unable to maintain a normal blood sugar (glucose) level.
Nearby Doctors
Dr. Bradley Barnett, MD is similar to these 3 Doctors near Tucson, AZ.
Tucson, AZ
Tucson, AZ
Tucson, AZ


text:
Another Vincent Longo first, Bi-Brow combines brow-filling powder and pomade in a single package. Designed to contour and shape the brows for a polished, feminine look. The non-greasy pomade tames brows and holds them in place all day as the powder adheres to hairs to build natural volume. A sheer tint leaves a soft, natural luster and rich color.
How to use: Dip a brush into the powder. Using short brushstrokes, begin at the highest point of the brow's natural arch and move along to the outer tip. Next, lightly fill the inner half of the brow using the same technique. Using a brow or lash comb, brush the brows to soften the powder and blend it into the brows to increase volume. Dip your finger into the pomade, gently dab it on the brows then comb the brows for direction.
- By Vincent Longo.
- Item #568261
Talc, Zink Stearate, Silica, Ethylhexyl Palmitate, Dimethicone, Mineral Oil, Lauroyl Lysine, Methylparaben, Propylparaben, Titanium Dioxide, Iron Oxides (CI 77491), Iron Oxides (CI 77492), CI 77499, Ultramarines, Mica.
Ingredients are subject to change at the manufacturer's discretion. For the most complete and up-to-date list of ingredients, refer to the product packaging.


text:
mau5trap artist, and owner of Kane Audio, a globally recognised sound-designer, producer, and studio engineer. Having been featured on the front page of Future Music Magazine, Computer Music, and provided sound-design for Moog, FXpansion, Steve Duda, Cakewalk, and many more, he's as technically skilled as he is creative.
As a writer & producer, his music has been heard globally in TV Shows, Films, and DJ sets from David Guetta, Deadmau5, Orbital, James Zabiela, to name a few, with international radio play from BBC Radio 1 & Sirius (Pete Tong), to Capital FM (Andi Durrant). He's engineered for several UK Top 40 artists recording vocals and instrumentation including Sian Evans (Kosheen), Chris Jones (Armada), and more. As a DJ, Dom has held residencies at some of the worlds most legendary clubs including Space Ibiza and Ministry Of Sound in London.
As a sound designer, the name Dom Kane has developed factory presets for synthesisers including FXpansion (D-CAM & Geist), Deadmau5 and Steve Duda (Nerve/Xfer), Ohm Force (Ohmicide), and the legendary hardware synth company Moog (Animoog, Little Phatty, Slim Phatty, Sub 37).
His sound banks and samples have appeared in studios worldwide via his own label Quantum Loops, and his singles and remixes have topped the Beatport Top 40 as well as having a full artist feature and front cover DVD in Future Music Magazine, and Computer Music Magazine. Ministry Of Sound TV released a worldwide exclusive preview of his "Syntax Error" across their networks, gaining over 10,000 views in just 48hrs on the MOS YouTube channel, and his music was featured in the League Of Legends global gaming competition with over 300,000 plays to just his music alone on YouTube.
Send me a note through the contact button above.
Gear highlights
- Custom built and GIK treated studio
- Audient AD/DA
- Event Opal Monitors
- Focal Monitors
- Yamaha Monitors
- Kyma Pacarana
- Moog
- Roland
- Korg
- Alesis
- Rode
- Bayerdynamic
- Emu
- Custom Valve Preamps
Genres I specialize in
Terms of Service
Turn around time varies depending on the project, but mix/masters usually 2-3 business days. Additional equipment and services are available upon request.
Reviews of Dom Kane
Very fast and professional and I am very happy with the results.
Dom is phenomenal at his work. Super happy with the Mix/ Master. Went above and beyond to accommodate my requests. Will defo work with him again... very soon!
I would highly recommend Dom to anyone. High professional with communication every step of the way.
Best sound designer and EDM mixer/producer that I've ever used!


text:
He was in on just about every play on defense as a safety, doing a good job when playing up in the box and stopping the run, and also making some good tackles down field.
He's got great closing speed and very good ball skills.
ScoutTV presents highlights of Carr in the win over Workman.
Twitter | Blog | Facebook | E-Mail |


text:
Для получения актуальных цен по гостинице Anemoessa Villa, пожалуйста, укажите интересующие вас даты и выполните поиск.
Anemoessa Villa is located near the beach in Santorini's Oia neighborhood and close to Naval Museum, Oia Castle, and Baxedes Beach. Additional area points of interest include Archaeological Museum and Museum of Prehistoric Thira.
Anemoessa Villa features a coffee shop/caf? and a bar/lounge. Room service is available. The hotel serves a complimentary buffet breakfast each morning in the breakfast area. Recreational amenities include an outdoor pool. Complimentary wireless Internet access is available in public areas. For a surcharge, the property offers a roundtrip airport shuttle (available on request). Tour/ticket assistance, secretarial services, and tour assistance are available. Guest parking is complimentary. Additional property amenities include a library, multilingual staff, and laundry facilities. A total renovation of this property was completed in March 2013.
Заезд: 14:00
Выезд: 11:00
Раннее заселение и поздний отъезд по возможности (услуга может быть платной).
Этажей: 2
Номеров: 13
|Описание номеров
|13 air conditioned guestrooms at Anemoessa Villa feature coffee/tea makers and complimentary bottled water. Rooms are all accessible via exterior corridors. Furnished balconies or patios offer sea or pool views. Beds come with pillowtop mattresses. At this 4 star hotel, accommodations include kitchenettes with refrigerators, kitchen linens, and cooking facilities. Bathrooms feature showers with handheld showerheads. They also offer complimentary toiletries and hair dryers. Wireless Internet access is complimentary. In addition to safes, guestrooms offer free local calls (restrictions may apply). Televisions have satellite channels. Rooms also include windows that open. Guests may request irons/ironing boards and wake up calls. Housekeeping is available daily.


text:
What Tools Do You Use?
Decorating By thelittlecakery Updated 21 Jul 2013 , 1:55pm by countrygirl318
AIm looking at fondant and gum paste modeling tools and wonder which brand/kind you use, have tried, absolutely hate and what you would recommend. I know a lot of people don't use tools, I'm not that confident to not use tools yet so any suggestions would be great!!!
AIf you want a sharp edge I use duff decorating tools and Wilton I love both of them
Quote by @%username% on %date%


text:
The Education Committee monitors the district's education system in its broadest sense. It acts as a reviewing body for curriculum proposals and district policies. The committee reviews state and federal legislations that impact the district and studies the research findings of professional education associations. It initiates analysis of new issues and is currently exploring methods for preparing students for the future.
Patrick O'Sullivan, Chair
Kate Murphy Peterson, Vice-Chair
Melissa Gracias, Board Representative
Denis Ryan, Board Representative
Rick Nogal, Ex Officio Member
Dr. Jim Gay, Superintendent, Ex Officio Member
Dr. Kim Dryier, Assistant Superintendent for Instruction
Dr. Stacey Gonzales, Director of Curriculum
Deborah Baker, Principal Sandburg
Eric Olsen, Principal Stagg
Robert Nolting, Principal Andrew
Michelle Etchason, Teachers' Association Representative
Sheli Thoss, Teachers' Association Representative
TBA, ESP Association Representative
Kevin Purcell, VJA Community Representative
Tammy Shoemaker, VJA Community Representative
Kathy Zwartz, VJA Community Representative
Dr. Mohamed Elnatour, CS Community Representative
Dr. Mitra Fallahi, CS Community Representative
Janet Donne, CS Community Representative
Zoe Kopanis, AAS Community Representative
Jill Fiscelli, AAS Community Representative
Debra Pletzke, AAS Community Representative


text:
About the Book
Features & Details
- Category Arts & Photography
Small Square, 7×7 in, 18×18 cm
30 Pages
- Publish Date Jan 09, 2013
See More
About the Creator
India Heron
Everything changes, even if we photograph something a thousand times it still won't re-create it the way we saw it. I photograph memories of moments that are happening but never forget to appreciate them whilst they do. I love photography but it can sometimes cause a longing for things that never can exist.
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text:
Club Events
We have over 100 clubs across Victoria who all hold various events throughout the year. This page is your guide to the special celebrations and events happening at your club, or in your area.
If you would like to advertise your club's event, be it a club concert, anniversary celebration or even a Summer School program, please email email@example.com
Bundoora Calisthenics Calling our to ladies over 26...
Bundoora Calisthenics Club is calling out for 2017 Masters participants. If you want to find out more call 0412247088 or check out www.bundooracalisthenicsclub.com.au.
Banyule Calisthenic Club's Closing Down Sale
Banyule Calisthenic Club is having a closing down sale on Saturday 11 February from 11am to 4pm.
Kingston Calisthenics Club Open Day
Kingston Calisthenics College are holding an Open Day at St Louis De Montfort's Primary School Hall, Aspendale on Saturday 11 February 2017. For more information visit www.kingstoncalisthenics.com.
Come and Try and meet Cali Bear at Avonde Calisthenic Club
Avonde Calisthenics are hosting a Come and Try event with Cali Bear on Saturday 11 February. For more information email firstname.lastname@example.com.