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The present invention relates to immuno-compatible hydrogel systems.
Immunoisolation and transplantation of primary or genetically engineered cells of allo- or xenogenic origin holds great potential to treat many hormone and enzyme deficiency disorders. The principle application of the technology has been the treatment of a wide variety of endocrine disease, including diabetes mellitus, hypoparathyroidism, dwarfism, central nervous system diseases, including Parkinson's, Alzheimer's, ALS, other genetic disorders including lysosomal storage disorders (LSDs), hemophilia as well as other conditions like kidney and/or liver failure and cancer.
The basic idea of microencapsulation is to entrap cells in a semi-permeable polymeric hydrogel and implant them into the body where, ideally, they remain undetected by the immune system for as long as possible. Often, the hydrogel alone is too permeable, so it is coated with a thin permeability-controlling shell. The most common type of microcapsule is the alginate-poly-L-lysine (PLL)-alginate (APA) capsule. An APA capsule consists of a calcium-alginate hydrogel core, surrounded by PLL (a polycation) and a final coating of alginate (a polyanion). The major advantages to using alginate are that it is processable at physiological conditions, and it does not interfere with cellular function.
However, the inconsistencies associated with alginate (a naturally occurring polysaccharide, composed of varying amounts of β-D-manuronic (M) and α-L-guluronic (G) acids, when isolated from different sources and purified by different procedures, is a major disadvantage. In terms of an immune response, alginate has been shown to contain variable amounts of inflammatory or immunogenic proteins, polyphenols and endotoxins. These compounds may cause fibrotic overgrowth around the capsule, leading to cell asphyxiation. In terms of mechanical strength, capsule failure after transplantation has been attributed to weakening of the calcium-alginate core caused by exchange of calcium for sodium in the body, followed by core swelling and rupture of the immuno-isolating outer shell. As well, alginate has recently been reported to degrade by oxidative-reductive and hydrolytic processes in the body, raising further concerns about long-term applications. At best, alginate varies with harvest location and harvesting methods, and requires substantial purification to be acceptable for human use.
To improve APA capsules, synthetic polymers have been utilized with varying degrees of success. The use of synthetic polymers permits manipulation to alter polymeric properties and avoids residual biological impurities found in naturally occurring polymers. A diversity of covalent modifications utilizing synthetic polymers have been used to improve the mechanical and chemical stability, permeability, and biocompatibility of APA microcapsules. In this regard, polymer-bound reactive groups have been utilized which are typically less toxic, for example, covalent cross-links throughout a linear pluronic polymer (a triblock copolymer of poly(ethylene glycol) and poly(propylene glycol)) hydrogel core, using Michael-type addition between pluronic chains having thiol and acrylate end groups have been used, as well as microcapsules that form covalent bonds through photodimerization of modified poly(allylamine) or PLL in the capsular membrane. The use of a reactive polyanion-bearing acetoacetate groups that form covalent crosslinks with poly-L-lysine has also been described.
However, there remains a need to provide an improved hydrogel system which overcomes or at least reduces the disadvantages of existing systems, for example, immunological incompatibility, including for example, undesirable binding to endogenous proteins. In particular, existing crosslinking systems tend to contain residual functional groups even after crosslinking, and these may subsequently bind proteins or undergo other undesirable reactions.
An immuno-compatible hydrogel system is provided herein comprising a crosslinked polymer matrix which is resistant to protein binding.
Accordingly, in one aspect of the present invention, a hydrogel system is provided comprising a covalently crosslinked polymer matrix, wherein said matrix is essentially resistant to protein binding.
In another aspect, a method of making an immunocompatible hydrogel system is provided. The method comprises the steps of:
These and other aspects of the invention will become apparent in the detailed description that follows and by reference to the figures.
FIG. 1 illustrates line profiles from confocal images of hydrogel capsules comprising varied compositions of polyanion and polycation; and
FIG. 2 graphically illustrates the extent of the lateral in-diffusion of dextrans of increasing molecular weight into hydrogel capsules according to an aspect of the invention.
An immuno-compatible hydrogel system is provided comprising a covalently crosslinked polymer matrix that essentially lacks protein binding sites, and is therefore essentially resistant to protein binding.
The term "hydrogel" generally refers to water soluble polymer systems capable of being gelled using biocompatible means such as divalent cation binding and thermal gellation, and includes, for example, calcium alginate, barium alginate, agarose and hydrogel systems such as those described in Prokop et al. (Adv Polym Sci 1998, 136, 1-51 and 53-73), the contents of which are incorporated herein by reference, for example alginate-cellulose sulphate hydrogel mixtures gelled in calcium chloride/polymethylene-co-guanidine.
The term "immuno-compatible" refers to hydrogel systems which do not induce an immune response on administration to a host, e.g. exhibit reduced binding with endogenous host proteins. Thus, the present hydrogel system lacks protein-binding sites, e.g. reactive sites capable of binding with surrounding proteins, within the crosslinked polymer matrix. Reactive groups (e.g. anhydrides, reactive esters (N-hydroxysuccinimidyl, pentafluorophenyl), azlactones) on the hydrogel system are converted into unreactive groups to reduce the possibility of undesired protein binding that could otherwise cause or contribute to a deleterious immune response to the hydrogel on transplant. The term "lacks" or "essentially lacks" is used herein to mean that the polymer matrix exhibits a negligible or insignificant amount of reactive protein binding sites, e.g. less than about 1% the original reactive protein binding sites remain.
The term "covalently crosslinked" as used herein with respect to a polymer matrix refers to the formation of covalent bonds between reactive polymers within a hydrogel which are stable in the presence of an ionic solution (e.g. a sodium chloride solution at a concentration of about 1-2 M), or at high pH levels, e.g. pH 12-13, such as in the presence of 0.1 N sodium hydroxide. This is in contrast to electrostatic interactions which are commonly labile in the presence of such ionic solutions, and at high pH.
The present hydrogel system may be prepared using a water soluble polymer capable of being gelled using biocompatible means such as divalent cation binding, for example, calcium alginate and barium alginate, and thermal gellation. Other high viscosity gel-forming polymers such as cellulose sulphate may be used instead of alginate, or together with alginate, to form the primary hydrogel core. Ionic gelling agents suitable for this purpose include calcium chloride or barium chloride.
The resulting gel, for example in the form of beads or capsules, may then be coated with a first biocompatible polyelectrolyte, such as a polycation, to form a hydrogel-polyelectrolyte complex, e.g. a sodium alginate-polycation solution. Suitable polycations for this purpose include those having a molecular weight and charge density suitable for electrostatic binding to the hydrogel, as well as a sufficient quantity of reactive groups, e.g. primary or secondary amines, to participate in a subsequent crosslinking step. The appropriate molecular weight of a first polyelectrolyte will depend on the nature of the hydrogel, including composition, concentration and pore size of the hydrogel, as well as on the nature of the second polyelectrolyte, including its molecular weight. Accordingly, suitable polycations for use as the first electrolyte include those having a molecular weight that permit their diffusion into the hydrogel core, for example, having a molecular weight in the range of about 1-200 kDa, preferably 2-100 kDa, such as 4-15 kDa, and 15-30 kDa, and 30-60 kDa, including homopolymers and copolymers based on monomers having primary amine groups such as aminoethyl methacrylate, aminopropylmethacrylamide, aminoethyl acrylate, allylamine, vinylamine and related monomers. It may be advantageous to use copolymers of amine-containing monomers with 25 to 75 mol % of uncharged hydrophilic comonomers such as hydroxyethyl methacrylate or hydroxypropylmethacrylamide, in order to reduce the positive charge density and thereby reduce inflammatory responses on implantation of the system into a host, such as by electrostatic binding of proteins. Other suitable polyamines include polymers such as poly-L-lysine, chitosan, polyornithine and polyethyleneimine.
The amount of the first polyelectrolyte appropriate for inclusion in the system is an amount that does not adversely affect the mechanical properties of the hydrogel core while being an amount that will result in sufficient covalent crosslinking on addition of a second reactive polyelectrolyte to the hydrogel, e.g. a polyelectrolyte that is capable of crosslinking with the first polyelectrolyte, for example, an amount that results in at least about a 1:1 stoichiometric functional group ratio between the first polyelectrolyte and the second polyelectrolyte. In one embodiment, a concentrated alginate hydrogel bead dispersion is exposed to three times its volume of a polycation as the first electrolyte at concentrations of about 0.02-1%, preferably 0.05 to 0.5, and most preferably 0.1 to 0.2 weight/vol % solution of polycation in aqueous saline or an aqueous solution containing both sodium chloride and calcium chloride. It is understood that these ratios may vary with the diameter and porosity of the hydrogel beads and possibly other factors such as the molecular weight, composition and amount of polyelectrolyte loaded onto the bead.
Following coating of the hydrogel with a first selected polyelectrolyte, e.g. a polycation, the hydrogel is then coated with a second polyelectrolyte, e.g. a polyanion, that is reactive with the first polyelectrolyte to covalently crosslink with the first polyelectrolyte. Suitable polyanions for use as the second polyelectrolyte (where the first polyelectrolyte is a polycation) have a molecular weight in the range of about 10 to 2000 kDa, more preferably in the range of 20 to about 1080 kDa, and most preferably in the range of 20 to about 500 kDa. Thus, examples of suitable polyanions include polyanions comprising reactive groups such as anhydride groups. Thus, suitable polyanions and precursors that become polyanions upon partial hydrolysis, include copolymers of maleic anhydride, cyclic anhydrides such as itaconic anhydride and citraconic anhydride, and linear anhydrides such as methacrylic anhydride, with comonomers that facilitate the polymerization thereof such as alkyl vinyl ethers, e.g. methyl vinyl ether and ethyl vinyl ether, and olefins such as ethylene and propylene. Copolymers of azlactones such as vinylazlactones, e.g. 2-vinyl-4,4′-dimethylazlactone with acrylic comonomers such as acrylic acid, methacrylic acid, and optionally a third neutral comonomer such as hydroxyethyl methacrylate or hydroxypropylmethacrylamide, are also appropriate polyanions, as are copolymers of N-acryloxysuccinimide, N-methacryloxysuccinimide or glycidyl methacrylate, with anionic comonomers such as acrylic comonomers and a third optional neutral comonomers.
The second polyelectrolyte may also be a neutral copolymer that is reactive with the first polyelectrolyte. Thus, neutral copolymers that are reactive with a polycation include, for example, those formed by copolymerization of electrophilic monomers selected from the classes of vinylazlactones, e.g. 2-vinyl-4,4′dimethylazlactone, succinimides, e.g. N-acryloxysuccinimide or N-methacryloxysuccinimide, and other strained ring-containing monomers such as glycidyl methacrylate, with neutral hydrophilic comonomers such as hydroxyethyl methacrylate, methoxypolyethyleneglycolmethacrylate, and acrylamide.
The amount of second polyelectrolyte appropriate for inclusion in the system is an amount that does not substantially affect the properties of the hydrogel while being an amount that results in sufficient covalent crosslinking with the first polyelectrolyte, for example, an amount in the range of about 3 times the volume of the hydrogel dispersion, with concentrations of second polyelectrolyte (or neutral equivalent) in the range of 0.01 to 1%, and preferably 0.03 to 0.3%. If polyanions of sufficiently high molecular weight, for example a molecular weight of at least about 250 kDa, or sufficiently high viscosity are used (for example, a viscosity of at least about 30 cps, and preferably higher), these polyanions may themselves serve as the hydrogel precursor as well as the polyanion.
As one of skill in the art will appreciate, the first polyelectrolyte may either be a suitable polycation or polyanion, and similarly, the second polyelectrolyte may either be a polyanion or a polycation. However, in order for covalent crosslinkages to occur, the first and second polyelectrolytes cannot both be polyanions or both be polycations. In cases where the first polyelectrolyte is a polyanion and the second polyelectrolyte is a polycation, it may be desirable to expose the hydrogel to an additional polyanion coating. In this regard, a small amount of a biocompatible polycation such as chitosan or a synthetic analog may be combined with the primary hydrogel in order that a single exposure to reactive polyanion would lead to the desired crosslinked network.
Cross-linking between the first and second polyelectrolytes may occur externally to form an outer shell on the hydrogel, e.g. the outer layer or surface of the hydrogel which may generally be about 1-100 micrometer in thickness, e.g. 1-50 micrometer in thickness. Some cross-linking between first and second polyelectrolytes may also occur within the hydrogel core, e.g. internal to the outer shell of the hydrogel, depending on the characteristics of each of the polyanion and polycation. For example, polyelectrolytes having a molecular weight of less than about 100 kDa, e.g. less than about 50 kDa, may also crosslink internally.
To provide a hydrogel system with enhanced mechanical properties, it may be desirable to prepare the hydrogel with additional first and second polyelectrolyte coatings as described above in order to yield an outer cross-linker shell of greater thickness, e.g. greater than 50 micrometers in thickness, higher density or greater cross-link density.
It may be desirable to provide a detectably labeled hydrogel system in order to render it trackable following its administration to a mammal, e.g. in the transplant of cells. In this regard, electrophilic reactive groups such as anhydride groups in the polyanion, may be labelled with a marker, such as a fluorescent marker, e.g. amino fluorescein, or may be modified to incorporate a chemical or biological modifier to otherwise enhance the function of the hydrogel system, for example to enhance the viability of encapsulated cells or the biocompatibility of the crosslinked network. Examples of suitable modifications include the incorporation of poly(ethylene glycol) groups by reaction of the initial polyanhydride with amino-poly(ethylene glycol), the incorporation of adhesion peptide sequences such as RGD, a tripeptide sequence consisting of arginine-glycine-aspartate, and the incorporation of spacers or other functionalities. Markers or modifiers may be added to the polyanion prior to its application to the hydrogel to engage in crosslinking.
In order to optimize the immuno-compatibility of the present crosslinked hydrogel, the hydrogel is further treated to eliminate residual reactive groups in the layers containing the second electrolyte, e.g. electrophilic groups of a final polyanion coating, or nucleophilic groups on a final polycation coating. The nature of this treatment will depend on the nature of the residual reactive group to be eliminated or converted to an unreactive group. Preferably, the treatment is a biocompatible treatment to ensure that the biocompatibility of the hydrogel system is not jeopardized. Thus, reactive electrophilic groups on polyanions may be converted on treatment with, for example, cytocompatible amines, such as aminoethanol or amino sugars, to form less reactive amide groups. Reactive anhydrides and other electrophilic reactive groups may also be converted to less reactive groups by hydrolysis. In this regard, a second reactive polyelectrolyte containing a large number of reactive groups may be exposed to a reaction to reduce the number of reactive entities and thereby yield a polyelectrolyte that retains the capacity to crosslink on a hydrogel coated with a first electrolyte, e.g. retain a sufficient number of reactive groups in order to undergo effective crosslinking. For example, a polyanion for use as the second polyelectrolyte may undergo partial hydrolysis in the presence of water to form a polyanion with a reduced number of reactive electrophilic groups, but still retaining a sufficient number of reactive electrophilic groups to undergo crosslinking with polycation deposited on a hydrogel. Residual electrophilic reactive groups that are not consumed in the crosslinking reaction with the polycation may subsequently hydrolyze on their own account, to form less reactive groups such as carboxylate anions.
The treatment or reaction to essentially eliminate or convert residual reactive groups on the hydrogel to less reactive groups may be conducted in the presence of facilitating agents. For example, the use of hydrolysis to convert residual reactive groups to less reactive groups may be facilitated by hydrolysis catalysts including certain enzymes, such as esterases, as well as tertiary amines.
In addition, the polymers (polyanions or polycations) may be modified to customize the properties of hydrogel system. For example, the polymer may be modified to introduce macromolecules having a desirable property, including anti-inflammatory cytokines (such as IL-10 and IL-2a), molecules which function to inhibit non-specific protein binding (e.g. amino-polyethylene glycol) or other regulatory proteins. This modification may be carried out prior to deposition of the reactive polyanion onto the polycation-coated calcium alginate beads (or deposition of polycation onto polyanion-coated beads). It may alternatively be carried out following deposition of polyanion onto the polycation-coated calcium alginate beads, provided that care is taken to ensure that sufficient reactive groups remain present on the crosslinked shell.
The present method, thus, yields a covalently crosslinked immuno-compatible hydrogel system with a reduced capacity to interact with proteins, e.g. a system that essentially lacks reactive protein binding sites (comprises less than 1% reactive protein binding sites) and, thus, is resistant to interactions with host proteins. The cross-linked polymer matrix also functions to stabilize the system, rendering it resistant to both chemical and mechanical challenges, thereby resulting in a hydrogel system having extended implant life in a host.
The present hydrogel system has widespread utility. At the outset, the cross-linked hydrogel system per se provides a stable, biocompatible, semi-permeable membrane. Among other utilities for such membranes, that would be well-known to those of skill in the art, an immuno-compatible crosslinked hydrogel membrane in accordance with the invention is useful in biomolecular separation techniques such as ion exchange and size exclusion chromatography. In this regard, it is noted that this system is not limited to the formation of beads and/or capsules, but may also be prepared as sheets of hydrogel by spin coating or deposition on a flat surface using a spreading knife, gelling using calcium chloride and crosslinking by exposure to the reactive polyamine. In this way, sheets consisting of covalently crosslinked polymer, with or without a target particle, may be prepared. In such applications, it is also possible to avoid use of alginate, and form crosslinked networks from only a bead, string or sheet of the reactive polyanion exposed to polycation.
The present hydrogel system is also useful as a biocompatible/immuno-compatible coating on devices for implant, including, for example, stents, catheters, other medical implants and the like.
Additionally, the present immuno-compatible hydrogel system is useful as an encapsulation system for use to transplant cells for the treatment of disease, such as lysosomal storage disease (LSD), diabetes, cancer or degenerative disease such as Parkinson's, and other conditions requiring cell transplant, or to deliver other encapsulated entities to a host, including for example, therapeutic agents, enzymes and hormones. In this regard, encapsulation of a target entity may be achieved by combining the entity with the hydrogel system during the polyelectrolyte coating steps. In this regard, it is noted that the present hydrogel system may be customized in order to provide a covalently crosslinked polymer network to retain the target entity, e.g. customized to have an average pore size that exceeds the size of the target.
Embodiments of the invention are described by reference to the following specific example which is not to be construed as limiting.
Materials: Poly(methyl vinyl ether-alt-maleic anhydride) (PMM), as purchased (20 kDa) from Scientific Polymer Products, Ontario, N.Y.; 1080 kDa from Sigma-Aldrich, Oakville, ON) were found to be partly hydrolyzed, and were hence heated in a vacuum oven at 140° C. for 5 days to reform to anhydride copolymer, PMM0, with less than 0.5% hydrolysis as determined by 1H-NMR. The fully hydrolyzed analog PMM100(216 kDa), 5-aminofluorescein (AF), poly(L-lysine hydrobromide) (PLL, 15-30 kDa and 40-60 kDa), tetramethylrhodamine isothiocyanate-conjugated bovine serum albumin (BSAr, 66 kDa, 1 mol TRITC per mol albumin), fluorescein isothiocyanate-conjugated dextran (10, 70, 150, 250 and 500 kDa), HEPES sodium salt, acetonitrile-d3 (99.96 atom% D), D2O (99.99 atom % D) and trypan blue stain (0.4% in 0.81% aqueous NaCl) were purchased from Sigma-Aldrich, Oakville, ON, and used as received. Sodium alginate (Pronova UP MVP batch no. FP-610-03) was purchased from Novamatrix, Norway. Sodium chloride and N,N-dimethylformamide (reagent, DMF) (Caledon Laboratories Ltd, ON), and calcium chloride (minimum 96% powder, anhydrous, Sigma-Aldrich, ON) were used as received. Sodium hydroxide and hydrochloric acid solutions were prepared from concentrates (Anachemia Chemical, Rouses Point, N.Y.) by diluting to 0.100 M or 1.000 M with deionized water.
PMM0 (100 mg) was dissolved in 1.0 mL of an acetonitrile-d3 / D2O (9:1 v/v) mixture in a screw-cap glass vial, forming a 10 w/v% solution. The vial was placed in an oven set to 60° C. for either 14.5 hrs (20k PMM0) or 17 hrs (1080k PMM0). Reaction times were determined by 1H-NMR to result in 50% hydrolysis, forming PMM50(20 kDa or 1080 kDa), respectively. At room temperature, 0.2 ml of the reaction mixture was diluted to 10 mL with 35 mM HEPES pH 7.8 buffered saline. The resulting slightly turbid solution was immediately agitated on a vortex mixer for 10-30 sec (at which point the solution was clear), and then quickly filtered (0.45 μm), to give a final aqueous coating solution containing 0.2 wt % PMM50. The complete dilution/agitation/filtration sequence was completed within 1 minute in order to minimize hydrolysis, and the solution was immediately used for coating. Coating solutions containing 0.2 wt % of the fluorescently labeled analog PMMf50, were prepared in the same way, except that 2 mol % AF (relative to total anhydride) was added at the beginning of the PMM0 hydrolysis in acetonitrile.
The degree of labeling for PMMf50 (20k and 1080k) was determined by dialyzing fully hydrolyzed PMMf100 against deionized water for one week, with daily water changes, using cellulose dialysis tubing with a molecular weight cut-off of 14,000 Da (Membra-cel, Viskase, Darien, Ill.) until the dialysate showed no signals for AF by UV-Vis spectroscopy. The polymer was then isolated by freeze-drying. The degree of labeling was found to be 1.03 and 0.88 mol % for PMMf50 of 20 kDa and 1080 kDa, respectively.
Preparation of FITC-labelled Poly(L-Lysine) (15-30 and 40-60 kDa).
FITC-labelled poly(L-lysine), PLLf, was prepared as described earlier (Mazumder et al. 2008. Biomacromolecules 9, 2292-2300. the relevant contents of which are incorporated herein by reference). For example, PLL (15-30 kDa, 100 mg, 0.48 mmol lysine.HBr units) was dissolved in 10 mL of 0.1M NaHCO3 buffer (pH 9) in a 20 mL glass vial. FITC (2.0 mg, 0.005 mmol) dissolved in 0.2 mL DMF was added to the PLL solution and the mixture was stirred for 90 min at 20° C. The resulting solution was adjusted to pH 7 with 1 M HCl and then dialysed for 5 days in deionized water using cellulose tubing (Spectrum Laboratories, 3.5 kDa MW cut-off) with daily water changes until the dialysate showed no absorbances for DMF or fluorescein. PLLf (15-30 kDa) was isolated by freeze-drying. Yield: 68.5 mg. The labelling degree was determined to be 1.05 mol % by UV-visible spectroscopy (73000 M−1cm−1) in 25 mM HEPES buffer (pH 7.4).
PLLf (40-60 kDa) was prepared in a similar manner from PLL (40-60 kDa, 202.8 mg, 0.97 mmol) in 18 mL of 0.1M NaHCO3 buffer (pH 9) and FITC (3.7 mg, 0.0095 mmol) in 0.37 mL DMF except that dialysis tubing with a 14 kDa MW cut-off (Membra-Cel, Viskase Corp., Darien, Ill.) was used. Yield: 148.2 mg. Labelling degree: 0.85%.
All capsules were prepared by a procedure described previously (Sun, 1988. Methods Enzymol. 137, p 575, the relevant contents of which are incorporated herein by reference). Briefly, a 1.0 wt % sodium alginate solution in aqueous saline was filtered (0.45 μm) and extruded through a 27 gauge needle at a liquid flow rate of 0.5 mL/min into a gellation bath containing 1.1 wt % calcium chloride and 0.45 wt % sodium chloride. The resulting calcium alginate beads were washed once with fresh gelling bath solution, followed by a saline wash. The alginate beads were then coated with PLL (15-30 or 40-60 kDa, both at 0.05 and 0.25 wt %) for 6 min. The resulting AP capsules were washed twice with saline, and coated with a 0.2% PMM50 (20 kDa or 1080 kDa) solution for 6 min at 10° C., followed by a saline wash. All coating and washing steps involved a 3:10 volume ratio of concentrated bead suspension to coating or saline wash solution. Table 1 identifies the various compositions prepared.
|Shell Thickness
|(full width at ½ height
|max height of
|of confocal microscopy line
|line profile
|A-PLL (wt %, MW)
|A-PLL(0.05%, 15-30k)
|A-PLL(0.05%, 15-30k)*
|A-PLL(0.25%, 15-30k)
|A-PLL(0.05%, 40-60k)
|A-PLL(0.25%, 40-60k)
|A-PLL(0.25%, 40-60k)
|All microcapsules were made from 1% alginate solutions, all coating solutions are in 0.9% NaCl, all PMM50 coating solutions were 0.2 wt %.
|*These are four-layer capsules A[PLL(0.05%, 15-30k)-PMM50]2, preparation details described below.
Analogous four-layer capsules were prepared by coating Ca-Alg cores with, in sequence, PLL (0.05%, 15-30 kDa) for 6 min, PMM50 (0.2%, 1080 kDa) solution for 4 min, PLL (0.05%, 15-30 kDa) solution for 6 mins, and PMM50 (0.2%, 1080 kDa) solution for 6 min, with two saline washes after each PLL coating and after the final PMM50 coating, and one saline wash after the first PMM50 coating.
Appropriate amounts of fully hydrolyzed poly(methyl vinyl ether-alt-maleic anhydride), PMM100 were dissolved in 35mM HEPES pH 7.8 buffered saline, to mimic the amount of carboxylic acid groups present in a 0.2% PMM solution with degrees of hydrolysis of 100, 80, 60, 50, 40 and 20%. The pH of these solutions were measured and used to generate a pH vs. % hydrolysis calibration curve.
Proton NMR spectra were obtained on a Bruker AV200 spectrometer. The pH of aqueous solutions was measured on a Corning 440 pH meter. Rates of hydrolyses in aqueous saline were measured using a PC-Titrate (Mandel Scientific) automatic titrator.
Capsules were examined by optical and fluorescence microscopy with an Olympus BX51 optical microscope fitted with a Q-Imaging Retiga EXi digital camera and ImagePro software. Capsules were also examined using a ZEISS LSM 510 confocal laser scanning microscope (CLSM) fitted with air-cooled Argon and HeNe lasers (LASOS; LGK 7628-1), and running LSM Image browser software (version 3.5). Images were further analyzed with ImageJ software to generate 10 pixel wide line profiles.
Capsule permeability was evaluated using fluorescein-labeled dextrans of 10, 70, 150, 250 and 500 kDa. For each dextran, approximately 20 AP-PMM50 capsules were placed on a microscope slide in the centre of a Teflon washer (1.5 cm diameter, 300 μm high), exposed to 200 μL of 0.1% dextran-FITC in saline, promptly covered with a glass cover slip and examined by fluorescence microscopy. This procedure, described recently (Gardner et al.) flattens and seals the top and bottom surfaces of the beads, transforming them into rounded cylinders that permit observation of lateral in-diffusion of the labeled dextran. Images were taken every minute for 20 min and the fluorescence intensities from the central ca. 20% of the beads, as well as from the surrounding continuous phase, were obtained using the ImagePro software. Reported intensity values are the ratio of intensity of the bead centre to the continuous phase, in order to minimize effects of photobleaching. These permeability measurements were carried out in triplicate for each MW of dextran.
In-diffusion of fluorescently-labelled dextran was also studied using a method involving a 24 h incubation of 0.2 g of microspheres in 0.0015% dextran solutions, followed by measurement of fluorescence intensity profiles across equatorial confocal microscopy sections using ImageJ software and 10 pixel wide line profiles.
Capsules made with a fluorescently-labeled PMMf were used to test for crosslinking. One drop of concentrated capsule suspension containing about 30 capsules was placed on a microscope slide and viewed by fluorescence microscopy. The supernatant was removed and immediately replaced with 2 drops of 1 M sodium citrate, a good calcium chelator, and the capsules were gently mixed. This process was repeated once. Subsequently, the supernatant was replaced with two drops of 0.1 M sodium hydroxide under gentle agitation, and the integrity of any remaining shells assessed qualitatively.
This procedure was carried out as described by Shen et al. J. Biomed. Mater. Res. B: Appl Biomater. 2009, the contents of which are incorporated herein by reference). Briefly, 0.1 mL of concentrated bead suspension was added to 1 mL of a 0.05% solution of rhodamine-labelled BSA (BSAr) in saline. After 24 h at room temperature, the capsules were washed five times with 1 mL saline for 2 min before examination by confocal microscopy for the presence and distribution of any residual BSAr.
Calcium alginate capsules coated with crosslinked shells formed by deposition of PLL (0.1%, 15-30k) and PMM50 (0.2%, 20k) have been implanted into immune-competent mice for 6 days. The capsules were incubated in Dulbecco's Modified Eagle's medium (DMEM) for 2 days. Prior to implantation the cell culture medium was exchanged for saline. The animals were treated in accordance with Canadian Institutional Animal Care guidelines. C57BL/6 mice (Charles River, Montreal QC) were anaesthetized with isofluorane (Anaquest, Mississauga, Ontario) before a suspension of 3 mL microcapsules in normal saline (total volume 5 mL) was implanted into the intraperitoneal cavity of mice under sterile conditions using a 20 gauge catheter (BD, Oakville, ON). After six days the mice were sacrificed and the capsules were retrieved and examined by phase contrast microscopy.
Microcapsules containing cells were similarly implanted. A C2C12 mouse myoblast cell suspension was mixed with the sterile alginate solution to a final cell concentration of 2 million cells/mL of alginate. The alginate/cell mixture was gelled and the resulting beads coated by sequential exposure to PLL (0.1%/15-30k or 0.05%/40-60k) and PMM50 (0.2%/20k). Following the final wash step, the microcapsules with cells were cultured in DMEM in a tissue culture incubator at 37° C. Prior to implantation the cell culture medium was exchanged for saline. The two types of capsules were implanted into immune-competent mice as described above.
Calcium alginate beads were coated with PLL (15-30 kDa) and then a fluorescently labeled analog to PMM50, PMMf50. Confocal microscopy was used to confirm that PMMf50 was bound to the capsule surface. With reference to Table 1, the full width at ½ height of the confocal microscopy line profiles is defined as the capsule thickness. All values have been calibrated to the same microscopy settings. Standard deviation (based on 6 values) is 2.5 μm or less for all width at ½ height values.
Line profiles from confocal images, as shown in FIG. 1, also suggest that only a small amount of PMMf50 (1080k) is actually bound to the surface. FIG. 1 illustrates line profiles of (a) [1] with reference to separate A-PLLf(0.05%, 15-30k) capsules (b) [3] with reference to separate A-PLLf(0.25%, 15-30k) capsules, (c) [4] with reference to separate A-PLLf(0.05%, 40-60k) capsules, (d) [5] and [6] with reference to separate A-PLLf(0.25%, 40-60k) capsules.
To confirm that the present shells involve covalent crosslinking in addition to the electrostatic linkages, the coated beads were exposed, in sequence to 1 M sodium citrate to chelate calcium and dissolve the calcium alginate hydrogel, and to 0.1 M sodium hydroxide to deprotonate PLL and break any electrostatic interactions. This method leaves only covalent crosslinks to preserve the structure of the shell. Capsules coated with PMMf100, a material unable to form covalent crosslinks, deflated upon extraction of calcium with citrate but still showed distinct shells consisting of an A-P-PMMf100 polyelectrolyte complex. When challenged with 0.1 M NaOH, the polyelectrolyte complex dissolved as PLL was deprotonated. Hollow shells resulted when AP-PMMf50 capsules were treated with citrate. Subsequent exposure to sodium hydroxide revealed the presence of intact shells or shell fragments, reflecting the presence of covalent crosslinking in the shell. Treatment of AP-PMMf50 4-layer capsules with citrate followed by sodium hydroxide revealed the presence of more pronounced final shells indicating increased thickness and mechanical strength. This multi-layer approach, thus, provides an alternative method for improving capsule properties.
A variety of AP-PMM50 capsules were prepared in order to optimize the strength as measured by chemical challenges (citrate and sodium hydroxide). In these experiments, the type and concentration of alginate, polycation, and the degree of hydrolysis of PMM were kept constant, while varying the molecular weight and concentrations of both poly-L-lysine and PMMf50, as well as the coating temperature. A-PLL(0.25%, 15-30 kDa)-PMM50(0.2%, 20 kDa) capsules were found to have the greatest resistance to the chemical challenges, while acceptable resistance to chemical challenges was observed with a greater range of molecular weights and concentrations.
Most cell encapsulations are carried out at 4-10° C. in order to protect the cells by lowering their metabolic rate. Hence, coating of AP beads with PMM50 was carried out at 10° C., as well as at room temperature. Many polymer properties, as well as the rates of hydrolysis and crosslinking, are temperature-dependent. The rate of hydrolysis should be lower at 10° C., allowing more time for coating and crosslinking, as well as leaving more anhydride groups to react with the amine groups of PLL. Measurements for both 20k and 1080k indicated that the rate of hydrolysis decreased slightly at 10° C. It was found that coating AP capsules with PMM50 (1080k) at 10° C. formed capsules that were no stronger than those made at room temperature. However, coating AP capsules with PMM50(20k) at 10° C. was found to improve the consistency and surface smoothness of the resulting AP-PMM50 capsules. Therefore, subsequent coatings were carried out at 10° C.
The thickness and density of the crosslinked shells depend on the ability of the polyelectrolytes to diffuse into the calcium alginate matrix. In particular, lower MW PLL would be expected to diffuse further into the primary calcium alginate beads, and would give rise to thicker shells, provided the reactive polyanion can follow the polycation. As a first step, FITC-labelled poly-L-lysine, PLLf, of two different molecular weights and at two different concentrations, was coated onto calcium alginate beads, and the resulting distributions were studied by confocal fluorescence microscopy. FIGS. 1(a) and (b) shows that the lower MW PLLf, 15-30 kDa, penetrates significantly into the beads, both at low coating concentrations of 0.05% and especially at higher coating concentration (0.25%). Accordingly, a higher molecular weight PLLf (40-60 kDa) was used and found to penetrate much less into the beads, at either of the two concentrations used (0.05%; and 0.25%, FIGS. 1(c) and (d).
AP beads made with either 15-30k or 40-60k PLL were coated with fluorescently-labeled PMMf to track the amount of PMMf bound and its distribution of the polyanion in the capsules. FIG. 1a and Table I indicate that high molecular weight PMMf50(1080 kDa) was largely restricted to the outer shell of the capsule. To test whether lower molecular weight PMMf50 could penetrate further into the beads, calcium alginate beads were exposed first to 0.25% PLL(15-30 kDa), which results in PLL being located throughout the bead, then to 0.2% PMMf50(20 kDa). The line profile from the confocal image, shown in FIG. 1b, and the width at ½ height stated in Table 1, suggests that while PMMf50(20k) does not penetrate significantly further into the bead than its higher MW analog, PMMf50(1080k), substantially more PMM50(20k) is able to bind to PLL. In the case of PLL(40-60k), the polycation is restricted to the surface but more is bound when higher coating concentrations are used (0.25% rather than 0.05%). The increased concentration of polycation at the surface allows for more PMM50 binding (as seen in FIG. 1d), resulting in slightly stronger shells.
To confirm that the thicker shell of the capsules of FIG. 1b still affords the permeability required for the cells' vitality in vivo, a kinetic permeability test as recently developed (Gardner et al. Langmuir, 2010) was used to measure the rate of in-diffusion of fluorescently labelled dextrans. FIG. 2 shows that 10 kDa dextran-FITC is able to diffuse rapidly into the capsules, reaching equilibrium within less than 10 minutes, suggesting that oxygen and small metabolites would rapidly pass through the exterior membrane. The 70 and 150 kDa dextrans diffuse at a much slower rate, and the 250 and 500 kDa dextrans are essentially excluded from the beads, suggesting that these crosslinked shells combine good permeability for low MW species with exclusion of high MW species, a desirable feature for materials designed for immuno-isolation. The apparent high rate of in-diffusion of the 150 kDa dextran is attributed to its extremely broad size distribution, which includes a significant amount of low MW chains.
One of the design points for the present PMM50 type capsules was that the rapid hydrolysis of residual anhydride groups prevents covalent binding of protein during the incubation step commonly used after cell encapsulation. To test this, A-PLL(0.25%, 15-30 kDa)-PMM50(0.2%, 20 kDa) beads were exposed to 0.05% BSAr for 24 hrs, followed by several saline washes. Subsequent confocal microscopy imaging showed no BSAr binding to the capsule shells, even with the detector gain set to its maximum. The absence of BSAr binding to the crosslinked shell indicates that no reactive anhydride groups remain on the capsule surface, and further that the hydrolyzed PMM does not bind BSAr electrostatically.
Implantation Studies of Empty Capsules Crosslinked with PLL/PMM50
Calcium alginate capsules coated with crosslinked shells formed by deposition of PLL (0.25%, 15-30k) and PMM50 (0.2%, 20k) were implanted into immune-competent mice for 6 days. After explantation, the recovered capsules were examined by phase contrast microscopy and showed minimal overgrowth, indicating the absence of significant immune response to these capsules.
Implantation Studies of Cell-Containing Capsules Crosslinked with PLL/PMM50
Microcapsules containing C2C12 mouse myoblast cell suspension and coated by sequential exposure to PLL (0.1%/15-30k or 0.05%/40-60k) and PMM50 (0.2%/20k) were implanted into immune-competent mice. After one week, the recovered capsules showed minimal overgrowth (less than about 5%), indicating immune-protection of the encapsulated cells from the hosts' immune system. Phase contrast microscopy images of the recovered C2C12-containing capsules, either A-PLL(0.1%/15-30k)-PMM(0.2%/20k) or A-PLL(0.05%/40-60k)-PMM(0.2%/20k), reveal that the majority of explanted capsules are clear (greater than about 80%) and do not show any overgrowth associated with immune recognition of the transplanted capsules by the host's immune system.
Copolymers of 2-vinyl-4,4′-dimethylazlactone (VDMA, TCI America) with methacrylic acid (MAA, Aldrich) were prepared and used as an alternate reactive polyanion for the formation of covalently crosslinked shells around calcium alginate beads. A study of the reactivity ratios of these two monomers revealed reactivity ratios of 1.42 for VDMA and 0.42 for MAA, indicating that in a copolymerization VDMA gets incorporated preferentially. To compensate for this tendency, the copolymerization was carried out in semi-batch fashion such as to maintain a comonomer ratio of roughly 35 mol % VDMA : 65 mol % MAA. This was done by starting with an initial comonomer ratio of 35 mol % VDMA to 65 mol % MAA, and adding sufficient VDMA at a decreasing rate during the copolymerization over the next two hours to approximately maintain this ratio. The copolymerizations were carried out in dimethyl sulfoxide (DMSO, anhydrous, Aldrich) as solvent at 70° C. for two hours, under nitrogen, and in the presence of one mol % azobis(isobutyronitrile) (Dupont) as radical initiator. The resulting polymer was isolated by precipitating the cooled reaction mixture into a ten-fold excess of cold diethyl ether. Optionally, small amounts of tetrahydrofuran, in amounts not exceeding the volume of DMSO, can be added to the reaction mixture to improve precipitation behavior. The composition of the resulting copolymer was determined by proton NMR in DMSO-d6 and found to be 60 mole % VDMA and 40 mole % MAA. The molecular weight of the polymer was determined to be 66 kDa by aqueous gel permeation chromatography analysis of a fully hydrolyzed version of the polymer.
Poly((methacrylic acid-co-2-vinyl-4,4′-dimethylazlactone) [PMV60], containing 40% methacrylic acid and 60% 2-vinyl-4,4′-dimethylazlactone, is water-soluble at neutral pH due to the 40% methacrylic acid content. To ensure rapid polymer dissolution while minimizing hydrolysis of the azlactone groups, solid PMV60 was dissolved in a small amount of DMSO prior to the addition of 35 mM HEPES buffer (pH 7.8) in saline. The aqueous PMV60 solution containing about 5% DMSO has a pH of 7.4, which is the pH at which coating is performed. Deposition of PMV60 on calcium alginate beads coated with PLL leads to electrostatic attraction followed by covalent crosslinking in a manner analogous to that described for PMM50. The reactive azlactone groups react with the amine groups of PLL to form amide crosslinks. Residual azlactone groups on the PMV60 were found by potentiometric and NMR analysis to hydrolyze within about one day to form carboxylic acids. Capsule preparation, coating and testing as well as implantation studies were conducted essentially as described in Example 1 for PMM-coated capsules.
Model capsules were formed by exposing calcium alginate beads to various concentrations of PLL (0.05 to 0.25% of 15-30k or 40-60k) and PMV60 (0.1 or 0.2%, 66k) to form shell-crosslinked capsules resistant to citrate and sodium hydroxide challenges.
Implantation Studies of Empty Capsules Crosslinked with PLL/PMV60
Calcium alginate capsules coated with crosslinked shells formed by deposition of PLL (0.1%/15-30k) and PMV60 (0.2%/66k) were implanted into immune-competent mice for 6 days as described above for PMM50-coated capsules. After explantation, the recovered capsules showed minimal overgrowth, indicating absence of significant immune response to these capsules.
Implantation Studies of Cell-Containing Capsules Crosslinked with PLL/PMV60
Calcium alginate capsules containing C2C12 mouse myoblast cells, and coated with crosslinked shells formed by sequential exposure to PLL (0.l%/15-30k) and PMV60 (0.2%/66k) were implanted into immuno-competent mice for one week as described above for cell-containing capsules coated with PMM50. After explantation, the recovered capsules showed minimal overgrowth, indicating that good immune-protection of the encapsulated cells from the host's immune system. Phase contrast microscopy images of C2C12-containing capsules formed using PLL 15-30k and coated with PMV60, explanted from the peritoneal cavity of immune competent mice after one week are clear and do not show any overgrowth associated with immune recognition of the transplanted capsules by the host's immune system.
A reactive polyanion formed by partially hydrolyzing poly(methyl vinyl ether-alt-maleic anhydride) was coated onto AP capsules to form a shell with covalent cross-links to the amino groups of PLL. Controlled hydrolysis of PMM0 was performed in ACN-d3/D2O and monitored by 1H NMR. At 50% hydrolysis PMM50 was diluted in a buffered saline solution, at which point hydrolysis was found to occur much more rapidly such that no anhydride groups would remain shortly after coating was completed. It was also shown that the distribution of the polyelectrolytes in the calcium alginate bead (amount bound, penetration depth into the hydrogel) depended on the MW of the PLL and PMM. The capsules formed with partially hydrolyzed PMM50 comprised shells that withstood challenge to sodium hydroxide, unlike those formed with the fully hydrolyzed PMM100, demonstrating that these shells were covalently crosslinked. During this process, it was found that higher concentrations PLL lead to stronger capsules that were better able to resist chemical challenges. Permeability studies showed that the covalently crosslinked shell formed around the AP-PMM capsules allowed for rapid in-diffusion of small molecules such as oxygen and metabolites while excluding larger MW species, such as immunoglobulins. The covalently crosslinked shell did not bind BSA, confirming that no remaining reactive anhydride groups are present following coating.
In addition, a reactive polyanion formed by semi-batch copolymerization of methacrylic acid with 2-vinyl-4,4′-dimethylazlactone, PMV60, was used in similar fashion to crosslink the outer layers of calcium alginate beads coated with PLL.
Both PMM50 and PMV60 crosslinked capsules, implanted into the peritoneal cavities of mice either with or without cells, showed little overgrowth following explantation after one week, indicating the successful isolation of the contained cells from the host's immune system.


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Sole Supports Plans Hohenwald, Tennessee Manufacturing Plant
The company will keep its current Lyle location but will expand to an existing 16,000 square foot facility in Hohenwald that the company will renovate. With this new facility, the company plans to expand its business and keep up with the growing market demand.
"Hohenwald was chosen as the home of our new facility due to its proximity to our Lyles location and the acquisition of a former textile manufacturing building that was well-suited for our needs," Sole Supports COO Ken Burks said. "We are very fortunate to find ourselves poised for growth and expansion and happily found all the attributes we need in the Hohenwald community to support our expected growth.
"We are excited to have the opportunity to grow and will continue developing and growing the Lyles operation as we develop the Hohenwald facility. The herculean efforts and support of the Tennessee Department of Economic and Community Development, Lewis County and Hohenwald City governments were tremendously important to influencing our decision to make Hohenwald the home of our expansion. Each have made the project easier and better than we could have on our own. We thank them and the citizens of Lewis county for rolling out the welcome mat for us so graciously," Burks added.
"It is great news to have Sole Supports expanding in Lewis County," Economic and Community Development Commissioner Randy Boyd said. "Over 9,400 Tennesseans are employed in medical equipment and supplies manufacturing and it is great to see that number grow with the help of Sole Supports. I want to thank Sole Supports for choosing to expand in our state and for creating valuable jobs for our Team Tennessee workforce."
"The city of Hohenwald is extremely pleased and appreciative to be able to welcome Dr. Ed Glaser and the fine people of Sole Supports to our community. The city has already established a close-knit relationship with the team from Sole Supports and feel that we are now a part of that team," Hohenwald Mayor Danny McKnight said. "During the past few months we have been able to work hand in hand with these people and consider them a part of the city of Hohenwald family. We would like to take this opportunity to officially welcome them to our city and county as not only a new and valued asset to our community but also embrace them as our new friends, neighbors and family."
"TVA and Meriwether Lewis Electric Cooperative congratulate Sole Supports on selecting Hohenwald, Tennessee," TVA Senior Vice President of Economic Development John Bradley said. "Attracting and retaining quality jobs and investments in our region is a fundamental part of TVA's mission of service. We are proud to partner with Tennessee Department of Economic and Community Development and the city of Hohenwald to further that mission and celebrate this announcement."
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subset: commoncrawl


text:
Louis Vuitton Red Epi Leather Cosmetic Pouch
This product is sold out
Louis Vuitton Red Epi Leather Cosmetic Pouch
In pre-loved/used condition. Visible usage, (see pictures). Exterior has no rips, tears or breaks in leather. Interior lining contains peeling/stains from usage (see pictures). No damages to shape/structure.
Ships as is.
Length 6.5" x Height 4" x Depth "2
Code #: Not legible.
All our items are authenticated by our professionals and will be shipped with care.
Majolie Designs established business in 1991, We certify that our products are 100% authentic or your money back guarantee. Feel free to contact us for more information or questions. All Sales Are Final.
(619) 405-2000


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|Copyright AUTO Insurance Quotes. All rights reserved.
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- 07 Oct, 2017 1 commit
Yonghong Song authored
The bpf sample program tracex6 is enhanced to use the new helper to read enabled/running time as well. Signed-off-by: Yonghong Song <firstname.lastname@example.com> Acked-by: Alexei Starovoitov <email@example.com> Acked-by: Daniel Borkmann <firstname.lastname@example.com> Signed-off-by: David S. Miller <email@example.com>
- 05 Jun, 2017 1 commit
Teng Qin authored
$ trace_event tests attaching BPF program to HW_CPU_CYCLES, SW_CPU_CLOCK, HW_CACHE_L1D and other events. It runs 'dd' in the background while bpf program collects user and kernel stack trace on counter overflow. User space expects to see sys_read and sys_write in the kernel stack. $ tracex6 tests reading of various perf counters from BPF program. Both tests were refactored to increase coverage and be more accurate. Signed-off-by: Teng Qin <email@example.com> Signed-off-by: Alexei Starovoitov <firstname.lastname@example.com> Signed-off-by: David S. Miller <email@example.com>
- 12 Aug, 2015 1 commit
Kaixu Xia authored
There are two improvements in this patch: 1. Fix the build warnings; 2. Add function read_trace_pipe() to print the result on the screen; Before this patch, we can get the result through /sys/kernel/de bug/tracing/trace_pipe and get nothing on the screen. By applying this patch, the result can be printed on the screen. $ ./tracex6 ... tracex6-705 [003] d..1 131.428593: : CPU-3 19981414 sshd-683 [000] d..1 131.428727: : CPU-0 221682321 sshd-683 [000] d..1 131.428821: : CPU-0 221808766 sshd-683 [000] d..1 131.428950: : CPU-0 221982984 sshd-683 [000] d..1 131.429045: : CPU-0 222111851 tracex6-705 [003] d..1 131.429168: : CPU-3 20757551 sshd-683 [000] d..1 131.429170: : CPU-0 222281240 sshd-683 [000] d..1 131.429261: : CPU-0 222403340 sshd-683 [000] d..1 131.429378: : CPU-0 222561024 ... Signed-off-by: Kaixu Xia <firstname.lastname@example.com> Acked-by: Alexei Starovoitov <firstname.lastname@example.com> Signed-off-by: David S. Miller <email@example.com>
- 10 Aug, 2015 1 commit
Kaixu Xia authored
This is a simple example and shows how to use the new ability to get the selected Hardware PMU counter value. Signed-off-by: Kaixu Xia <firstname.lastname@example.com> Signed-off-by: David S. Miller <email@example.com>


text:
Pumpkin Spice Artisan Soap
Dare to be basic and pumpkin spiced, but still be extra with purple swirls, glitter gems and crystals, and all the glitter.
Scent: Pumpkin Spice: Cinnamon, Ginger, Nutmeg, Pumpkin, Clove, Vanilla. Contains natural essential oils, including real cinnamon, clove, and ginger.
Oils Used: olive, coconut, castor, and sustainable palm oils, shea butter
Color: activated charcoal*, mica and biodegradable eco glitter ✨
Also contains glycerin soap decorative top.
Also available as a candle, diffuser and spray.
*charcoal is great at cleaning oil, but thus can overcleanse dry or sensitive skin. If that's a concern, skip the charcoal and opt for another soap like Castile or Aloe.
Free of parabens, detergents, and sodium lauryl sulfate and sodium laureth sulfate, which can dry out sensitive skin.
4 oz bar wrapped in a recycled paper bag, plastic free. Each bar is hand cut, so slight variations are possible. Roughly 1"x3.5"x4" per bar. This style lasts longer than glycerin soap and storebought name-brand soaps! Keep dry to lengthen the life of your bar.


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The International Conference on "Role of media in democracy" was hosted by Xavier School of Communication (XCOMM). Mr. Siddarth Varadarajan, the founder of "thewire.in" was the chief guest and Mr. Baijayant Panda, Honourable Member of Parliament was the guest of honor, Fr. Dr. Paul Fernandes SJ, EX-Vice Chancellor, Xavier University, Fr. Lourduraj Ignacimuthu SJ, Dean, Xavier School of Communications (XCOMM), and Ms. Jagi Mangat Panda, Managing Director of OTV were present at the inaugural session.
International Conference in Collaboration with OTV. The theme was Role of Media in Democracy: 25th & 26th November, 2016 (Friday and Saturday)
- November 25, 2016


text:
Canadian Hospital Specialties is a local manufacturer in Oakville and a national specialty distributor of medical and surgical products focused on strategic partnerships to enhance the experience of our customers.
For over 50 years our clinical sales and marketing expertise coupled with our continued commitment to invest in new technologies brings value to supplier partners and customers alike. Our partnerships are based on shared values and mutual respect, essential elements to ensure long term success.


text:
Price: $30.00
Quantity: 1 available
Book Condition: Good+ with No dust jacket as issued
Text is clean. Cover shows wear, uneven tanning to front cover. Appears to be a monastery library copy, but with no marks beyond front free endpaper. Previous (private) owner name stamped on front free endpaper. Black cloth with blind stamped decorations and gilt cover lettering. Corners bumped/rubbed. ; This volume reprints the text of several lectures given by Bishop McQuaid in defense of Catholic education at a time, when the establishment of a parochial school system was running against public opinion in New York.
Title: Christian Free Schools The Right of Parents to Provide Religious Education for Their Children without Let or Hindrance
Categories: American Catholicism,
Edition: First Edition
Publisher: Rochester, NY, Union & Advertiser Press: 1892
Binding: Hardcover
Book Condition: Good+ with No dust jacket as issued
Item: 1.00 Item
Seller ID: 25183


text: Hi, I am looking to purchase 10 LLLL.com(s) in bulk for for NET $2.5k Domains must meet the following requirements. 1. All LLLL.com Only. 2. Minimum 6 months to expire. 3. Minimum 5 or more domains must be CHIP i.e. Should not contain (A E I O U V) 4. Remaining 5 or fewer domains should not contain (Q W Y U J K Z X V) If the above requirements are met and you would like to liquidate a part of LLLL.com portfolio Please PM Me. I will pay $2500 net - All other fees are borne by the seller. Thank You Raj RajDomains


text:
Wabash to Move Away From Sheepskin Diplomas After 187 Years
Over time at Wabash, traditions change, some fade away, and some new ones crop up. We all know that these traditions are not fixed, but that does not make it any less unfortunate when one of the College's longest standing traditions fades away. It appears that will be the case when commencement rolls around for the Class of 2020, who will be the first class not to receive their diploma printed on sheepskin parchment.
The news that the College would be moving away from the diplomas first broke on October 2nd, when The Wabash Commentary posted the story on their Facebook page. The news was later confirmed when Wabash President Gregory Hess attended the Student Senate meeting on October 8th. During the meeting, Hess confirmed that diplomas would no longer be printed on sheepskin and took questions on the sheepskins and a number of other issues. Hess pointed primarily to "deteriorating quality" and "inconsistent production" on the sheepskin diplomas for the change, saying the quality recently was, "beneath the great tradition of this College." However, Hess acknowledged that the communication of the decision was subpar. He said, "I will own that communication here could have been a heck of a lot better."
When the forum was opened up for students speak, many expressed frustration with the decision, saying that they believed it was motivated by a desire to cut costs. Hess denied this allegation, though he acknowledged that costs on the sheepskins were high and continued to rise. Many other students expressed their frustration with the way this decision was communicated and with similar breakdowns in communication in other decisions that the College has made recently. Some students cited housing changes a couple of years ago, which were made public in an article for The Bachelor, and a general lack of perceived communication from the administration as evidence that the administration was neglecting student opinions.
Hess repeatedly pledged during the meeting to work to improve communication with the student body, offering to come to Student Senate meetings more often and saying that he was open to ideas on how he can best hear student opinions in the future.
The news about the College's move away from sheepskin diplomas attracted interest from many prominent publications. In addition to The IndyStar, the story was published in The Chicago Tribune, The Washington Post, and The New York Times. The decision from Wabash leaves Virginia Military Institute as the only remaining college in the United States which still uses sheepskin for their diplomas.
The College is still considering exactly how to move forward with the diplomas, and a committee to determine what the next step will be has been formed, including student representatives. However, it appears clear that the sheepskin diplomas are now a thing of the past.


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Heading to Vietnam? or already there?, then check out some of these foods.
Mi Quang
A rice noodle dish, with a source of protein chicken/beef/pork. Sometimes with fish or seafood. Seasoned with turmeric, peanuts, chilli and lime. Served with sesame toast and salad.
Add your toast and salad into the soup. Let it soak up the juice and enjoy.
My advice don't wear white! It can splatter.
Seriously loved this dish. Yummy! With the amazing price of £1.15.
Banh Mi
It is a Vietnamese style baguette with filling. Usually stuffed with meat, pate, pickled veg and chilli.
Another amazing price of 83p per baguette, makes a perfect snack or on-the-go lunch. Of courses prices vary depending on vendor.
They sometimes have an array of fillings available, beef and cheese, bo la lot, nem nuong etc.
Com Tam
A very popular dish. Some restaurants have their own style of this dish. But it means broken rice. And originally served with pork but many people offer different meats. The main seasoning is a chilli fish sauce. Not to fear, the sauce isn't too potent for those none fish eaters, like myself. And I very much enjoy this, I probably eat this every 3 days. £1.16 for this one in the picture. It is usually accompanied by soup and green tea. Amazing!
Guys I often post on my instagram first, so if you wish to be the first to know then follow me. And as usual I aim to upload Monday/Thurs/Fri, until then take care. And as always, comments are welcomed and a like would be appreciated.
Disclaimer: Please not that any information displayed is from my research and may differ to other sources. In addition, please do not confuse my opinions with factual data. Photography displayed are either my own or from a paid subscription; and are not to be copied without permission .


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- 2 Onions - chopped
- 8 garlic cloves
- 3 large ripe tomatoes (peeled and Chopped)
- 1 large spoon tomato paste kunserva
- Olive Oil
- 100 ml white wine
- 6 small Fish (you can use heads and tails of bigger fish)
- 1 lemon
- Cup of rice - Optional
- Fresh mint
- Majoram (merqtux)
- Salt
Method:Slowly fry the chopped onions in oil.
Add the garlic, the mint, the marjoram, the tomatoes and the tomato paste.
Cook slowly for about 5 - 10 minutes
Add the fish, the wine and the water (with some salt added) and cook for 30 minutes.
Towards the end of the cooking time you can add the rice.
The aljotta is not meant to have a thick or heavy consistency but should look more like a fish broth.
Difficulty: Easy
Over the years i have tried many variants of this recipe – different types of fish can be used and I have tasted some with shellfish with vongole added. Rice is sometimes added to turn this dish into a full meal.


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The Kind Of Direction Our Film Industry Should Take And Why
Long gone are the Maula Jutt days (thankfully) and all that over the top Punjabi acting and that melodramatic action sequences that made the Movie World in Pakistan basically a joke. No one went to the theaters anymore. Thankfully we have successfully transitioned out of that phase in these last few years. Especially with the introduction of international cinemas and quality movies.
Along with that our own film industry took the necessary changes it could to improve itself and it did that successfully as we are finally producing movies worth-watching and ones that don't use a yellow filtered camera from the eighties. Finally people want to actually go to the cinema halls with family and friends and it looks like at last the 'going to the movies' business has been revived.
But, usually the good movies that have been coming out lately have been torn between, either about war or again following the age old formula of a typical Bollywood blockbuster that has action, comedy and romance all in one with a confirmed item number.
The kind of direction that our film industry needs to take, however, should be to develop it's own niche, and not limit that to just small independent films. There should be more creative freedom and a dynamic pool of stories. The actors and actresses should be diverse and not just limited to being conventionally beautiful. Stories should come out from all around Pakistan and new aspiring writers and directors should be encouraged so we can explore the realms of our strength and to find out where cinema could take us.
Let us not loose the audience that we have created. Lets aid to the expansion of the horizons of story telling so that our movies can lead to an international standard level and not just end up becoming a Bollywood rip off.


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Farmhouse Landscaping
Collection by JunkStyle Home
Shade Loving Flowers, Bushes, and Groundcover That Thrive Under Trees
12 Beautiful Hanging Planter Ideas
One of my favorite features in my garden is all of the hanging planters. If you ask me, a hanging planter just completes the space. Plus, these hanging planter ideas will allow you to maximize the space on your porch or patio so more plants are always welcome!
DIY Craft Projects using Old Vintage Windows Doors - Trash to Treasure - Architectural Salvage
recycled window frames mini green house
10 Enchanting Country Gardens That'll Charm Your Socks Off - Wide Open Country
Whether you live on 40 acres, or you're 40-stories high, you're sure to be enchanted by these country gardens.
Home and Garden DIY Ideas
Old wash tub planters More
SylviaS VintagE DaughteR
I REALLY am my mother's daughter...
Building a Plant Obelisk
Create a year-round garden accent with this stylish plant obelisk. Whether supporting vines or left unadorned, it adds a vertical design element to your garden.
<3 Rustirc angel wire, wood sticks, pipe cleaner,
New DIY Garden Rock Ideas
Projects with rocks are so diverse that they always bring a great natural beautiful element to where ever they are. You can bring this natural look down to your backyard simply using a collection of different rocks you can bring about a beautiful and natural aura to your garden, and guess what? These projects cost you virtually nothing at all! Below we have compiled a few ides which we think would be a beautiful addition to any garden, enjoy.
Leichte und einfache Bastelideen für DIY-Holzpaletten
15+ Awesome Shade Garden Ideas - Go Travels Plan
Some plants might be affected by air pollution while some aren't. Aromatic plants add a distinctive touch to a garden, developing a feeling of romance and a unique type of beauty.


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Current Version
No reviews found.
Before 1.1
Excellent solution!!
On the main page I'm using Page List+ to list all "event" page types with a thumbview template applied to pull the image from the single event block.
But.... every time the cache was cleared Page List+ presented me with an error, yet still pulled in the proper image:
Warning: imagejpeg(): Unable to open '/files/cache/74eb80b76bc556569315e720cd633629.com/1423846197830308/picture?type=large' for writing: No such file or directory in /updates/concrete188.8.131.52_updater/concrete/core/helpers/image.php on line 207
By installing Cache Filler and using Flex Job Scheduler I was able to run Cache Filler soon after every cache dump, therefore speeding up the render of those pages and getting the error out of the way!
This is the line I used to include the specific page in config/site.php:
define('CACHE_FILLER_PAGES', '/events/');
1.1 -
Really useful - should be in the core
The issue I had is that when the cache was cleared (in order to pick up updates in the data feed), the first time each page was then visited, it would load very slowly whilst it was cached again. I'd run into this problem a bit on other sites, but it was much more of an issue on this one due to volume of data.
So - this add-on was an absolute god-send. By adding in a job to run at the same time as when the cache is cleared, it completely fixed the issue.
Great work JohntheFish, and thank you for making this a freebie.


text: SKU 5022 Length Width Height Availability Pork Chop, Smoked These fully cooked pork chops are full of flavor! You'll receive 1 Smoked Pork Chop per package. Be the first to review this product Approx. $10.49 base price: $10.49 per 1 lb Qty: Add to cart Email a friend Question about product? Add to compare list Add to wishlist DetailsExisting reviews Try the deep, rich hickory smoked flavor of our Smoked Pork Chops! They are are fully cooked - just warm them up or brown them before serving on the stove, in the oven or on the grill. Each package contains 1 Smoked Pork Chop, approximately .80-1.25 lbs per package. The advertised price is an estimate based on exactly 1.00 lb. The final price will vary based on the actual weight of your package. All of our pigs here at John Henry's are fed a mix of corn, oats, and alfalfa to keep them happy and healthy. They are, locally raised, no hormones and no antibiotics. Our fields and feed are never exposed to harsh chemicals or pesticides. Ingredients: Pork, Water, Potassium lactate, Salt, Sugar, Sodium phosphate, Sodium Diacetate, Sodium Erythorbate, Sodium Nitrate Be the first to review this product Customers who bought this item also bought Ground Round - 1 lb pkg $9.39 Boneless/Skinless Chicken Breast - 2/pk $14.99 Bnls/Skls Chicken Thighs - 4/pk. $6.99 Hickory Smoked Bacon $10.99 Ground Chuck - 1 lb pkg $8.39 Pork Chops, Bone In 2/pk $8.39 < >


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Going off the grid is not simply for the survivalist in the wilderness of Alaska, or the increasingly independent household that left the big city for the more remote (and simpler) life in Salem OR. Going green, and living off the grid is no longer simply for the homestead family, nor the hermit isolating himself from the crowds. Sun getting photovoltaic panels are on roofings, in backyards, on remote jobsites, even campgrounds. You'll find them in the big city, town, in energy conscious communities, in residential, commercial, even commercial locations. The benefits are numerous, from assisting save the world and it's resources, to getting your independence from utility business. Not to discuss the best part - conserving a lots of money by significantly reducing, even completely eliminating the power costs.
Lastly, you ought to be comfortable with your handyman 's pricing being sensible and reasonable. Don't necessarily select a handyman based on the lowest cost, but rather try to find a reasonable price for the best work. And speaking of rates, make certain you understand the payment policy. Is a deposit needed? What are the techniques of payment accepted? When is the work anticipated to be completed?
deck builders needs to be specialists in developing both stairs and ramps and are a great resource for these types of jobs if you require somebody else to build them. If you https://trevorjjzt741.page.tl/House-Enhancement-Pointers-%26-Tricks.htm don't feel like developing it yourself, they can likewise be an excellent resource for getting a storage shed constructed as well. , if they are uneasy with building this type of structure they will still understand a lot of builders and specialists that may enjoy to do the work..
Opportunities are that if you had adequate structure abilities to develop your shed you have enough to tackle some easy strategies to construct a set of stairs or a tough ramp. Then you will require to figure out if you have actually the ability set to construct these essential devices, if you bought or assembled your storage unit.
After speaking with your possible remodeling contractors, and receiving some quotes from each of the companies, you need to have the ability to whittle down your option down to a couple of companies. Have a look at all the info you have and discover the ideal company for your job. Feel complimentary to give the company a ring and get the job officially began when you have actually effectively made your choice. You can kick back and unwind once they start!
Before working with a specialist you may want to hire a designer to produce the design that remains in line with what you want. Nevertheless if you desire to keep your costs as low as possible then you can bypass a designer and can choose for yourself what you desire the project to be and what elements it will need to consist of. It is also essential for you to make up a budget for yourself so you will understand exactly just how much you can and can not pay for to invest.
It would constantly be a really great concept for you to keep those things in mind if you would like to get San Antonio cooking area renovation. When you are already in a point of doing the primary thing, use them out. You'll be surprise about the great advantage that it might deliver for you.


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As Moscow is set ablaze by the retreating Russians, the Rostovs flee their estate, taking wounded soldiers with them, and unbeknownst to them, also Andrei. Pierre, dressed as a peasant,…
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I mentioned that I had to cut so as to send an email to this guy the phone call brief. She began to say, " Remember. . . " but I anticipated her instructions and promised her I would get his telephone number( lesson learned from the Cheesecake Factory fiasco) . I'd give him mine. Brooke just laughed, and we hung up.
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Say" I know a brand of cereal fewer calories" upon visiting a box of cereal in his cart, or" How do you think organic food may make a difference in my diet? " If you visit packs of vegetables and fruits in his things. Offering and accepting help can be a fun way. Hold a product you can use for flirting. The area where condoms are offered is a fantastic place to await a random cute guy. You can hold a box pretend like you are still deciding which odor and size that you want to buy and to grab his attention. Who knows, he may even provide some help to you. Whenyou're attempting to reach for a product on the shelf that is lower bend beside him. The movement alone already triggers a man's creativity. Letting him see some skin might force him to present himself and request your number then. Proceed to the magazine section and navigate through fitness magazines. When you find a guy already looking to purchase, this movement works.
What's the question? Well, why would a man who has dated women that are both positive and negative decide not to pursue a fantastic girl? Scared? If that's the case, scared of what? Total of lies? A backpage reviews for escorts Richmond PE boy in great man( sheep's) clothing? Well, in case you have time that I truly appreciate your response and insight.
Then, in the Backpage escorts over 50 Alma's Richmond PE instagram models hookers of this sitcom, that He watches me dwell from above, my daddy's friend was sent to the same rodeo, shortly spotting my" boyfriend" I desired nothing more than to hide from the ladies' room for the whole evening, but rather, I had to explain why this man wouldn't be joining us this summer out at the cottage.
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Continue When they email you to filter, filter them. As I've mentioned before, use. Be fully merciless. You're not doing. If you give an inch, these men are going to take a mile. They would have no hesitation in using and abusing you.
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Write down on that paper what you would look like. This description would be placed in the top spot on the newspaper. Then go to number two on the list on the notepad paper which could be, by way of example, a manner in which you presented yourself that would be less appealing than the description in the number one spot.
If you have seen the movie Sex and the City, I can only imagine that is what Carrie needs of felt like when it was determined that Big and her were making the big move to the infamous" M" word. Where she wore some of the most exquisite wedding dresses you had laid your eyes 32, on top of it all, she was given the wonderful photo shoot Vogue! She is on top of the world, what she's experiencing is what would be listed in the books as the supreme success.
Heexcels at intimidating his rivals to the ground and's a shark that is dominating. He tends to violate people constantly, and verges on being too aggressive with his friends. He is no stranger to pub fights and has anger problems.
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Find the Ideal Love Partner: Tips at the beginning stages of this manual that was relationship and how to Filter your Matches, we talked in length about the aspect of going on a date that was horrible with a person but whom you've nothing in common. While you may not be planning on dates in this specific instant, and are likely at the best military dating apps stages of progressively searching for a love match and building from there, it is also important to be mindful of who you socialize with; some games will just click, while some will expect a bunch of work.
Do not wait for everything to be perfect. Remember it's not simply you, there are lots of various other guys online as well attempting to get her out on a day. If you don't pull the trigger, this might inform a woman that you are not the type of person she's looking. You are doing fantastic currently If you are having a hard time asking her out or otherwise certain exactly how to ask her out as often all it takes is just one mistake in a message, as well as you lose the woman. Simply duplicate and paste this" probably we need to proceed this conversation /banter over a drink /coffee" or" allow's meet for coffee /drink in some cases exactly how does that Richmond PE only women dating apps" and gradually, you can compose your message. My job is to get your foot one the door as well as in time, your confidence will expand the more days you go to.
I also feel that my Twin Flame understood that I am the reason why he hadn't found what he was looking for in previous connections- - it's wonderful how Twin Flames get entitled for their own celestial partner- - he has this natural response to always desiring to protect me. To keeping me secure, he is attracted- - and he does.
I'd noticed that all these smart, accomplished competent people had an unusually hard time when it came to dating and relationships, when I was an advisor at Harvard a few years back. And I noticed that my college classmates were early, stuck in bad relationships or perennially single.
Are You Currently Portraying the Real You? Men always bail and if you get to a first meeting point, you might not be portraying yourself in a real transsexual escorts backpage Richmond PE. Go back through your profile and correct some announcements that you really don't reflect whoyou're. You stay because you truly hate dogs, especially if you said you loved them on your profile and then can't appear in a dog park.
Along with the large mistake most men make, is that they believe that they can find out a few lines and a couple of tricks, and that everything then will be hunky- dory. . . that getting the woman is the point where the answer lies. In reality, many men fantasize about being successful with girls. They envision Richmond Prince Edward Island backpage escorts banned up in clubs and bars, saying acouple'magic' words, and'bingo! ' The girl would then happily go back for a night of passion! ! And some guys really play out these dreams exaggerating any victory they have with women. Richmond phillipino prostitutes to the point of making up stories of success and pretending. The" We did everything! " Stories Richmond backpage 40 dollars escorts away the game. Rather than everything, substitute in you and the word NOTHING would then have a more accurate description of what happens in these stories that are make- believe! Men envision relationships in their minds: Imagining a beautiful woman will one day fall in love and that they will be able to keep her psychologically and physically satisfied for as long as they desire.
First, consider this as the" What I'm looking to get" section. So that you know what an perfect match resembles to you by this point, you should have outlined your relationship goals. You ought to know exactly whatyou're looking also and for what you are not searching for.
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Sports clubs: A ticket to the baseball or football game can be expensive, but if you are aware that you are both interested in sports, exploring your choices here is made so much easier for those who" go Dutch" . You enjoy watching or tennis basketball. Do your first date while cheering on your team.
When you have time or when you want to, the wonderful thing about online dating is that you obtain to message individuals. You do not need to offer a spontaneous reaction to everything they say to you. Instead, you can actually think and also take some time regarding what they are saying and also what you intend to say in backpage escorts blocked Richmond Prince Edward Island. This way you can see to ityou're really stating what you want them to listen to.
When speaking to other women, it seems that is the most barbarous is that the" single season" . For whatever reason it comes across that ifyou're one of those who are at the section that is single; you have this curse that is currently hanging over your head that just prince magical can break.
Each time I attempt to withstand the Twin Flame connection; sometimes I become doubtful- - that I feel disconnected from the entire world and myself and occasionally I obstruct his energy or thoughts of him. The refusal of the Twin Flame connection in any way pushes me to feel empty and numb.
If if a girlfriend sends you a message such as" You're completely different from the others I have met! " , rather than answering her the usual" You too, my love, I love you so much! " , you'll answer her" Yes, I must admit I'm somewhat autistic" ( smiley) , or a simple and simple" thank you" . I can GUARANTEE you that you will bring her more, because she'll think lack of favorable" affection" that will push her to search for even more in you.
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The second in our series on emerging artists in Taranaki, Waitara-based painter Rubi Riot elaborates on the stories behind her colourful, layered paintings: of music, myth, magic, love and house fires.
Rubi Riot mentions North Taranaki Arts Society (The Gables) — anyone can join and show work in regular exhibitions.
-39.056159 174.077697


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If I had to choose my favorite berry of all time, it would be blueberries…for sure. I'm so lucky that we can getting them locally, practically in my backyard! Recently, … Read More
Pumpkin Cheesecake Protein Bars
Pumpkin Cheesecake. Does it get more fall than that? Only what's different about these Pumpkin Cheesecake Protein Bars is that there is no cream cheese in them at all! They … Read More
Cheesecake Protein Bars
I've fallen in love with these healthy cheesecake protein bars. I think there's a good chance you will too. How do you get the taste of cheesecake, but no cream … Read More


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Create an Account - Increase your productivity, customize your experience, and engage in information you care about.
City Council allocated Measure O funding to develop a Senior Strategic Plan that will allow the City of Ventura to:
GreenPlay, LLC, a nationally-known parks and recreation management consulting firm, has been hired by the City of Ventura to develop the Senior Strategic Plan.
You're invited to share feedback and input on the draft version of the Senior Strategic Plan developed by GreenPlay from Wednesday, January 29 through Wednesday, February 12, 2020. All comments and feedback will be provided to GreenPlay before they finalize the version of the Plan that will be presented to City Council on March 16, 2020.
UPDATE: Due to the COVID-19 pandemic, the City Council presentation of the final version of the Senior Strategic Plan will be rescheduled.
Please send comments and feedback to:
Emily Fox
Community Partnerships Manager
firstname.lastname@example.com; 805-658-4793
Printed versions of the draft Plan are also available for review at the following city facilities:
During this Phase 4 the consultant shared the Key Issues, organized into four Key Themes, that were identified during the strategic planning process and included in the draft version of the Senior Strategic Plan. In addition, the goals and strategies to address these needs and priorities were provided with the opportunity for community members to share feedback.
In Phase 3 the consultant shared their findings from the community engagement meetings held during Phase 1, the senior needs assessment survey conducted during Phase 2, and their independent research and analysis. They introduced the draft Key Issues Matrix that identified priorities and needs for older adults in the City of Ventura along with their initial recommendations on how each could be addressed.
Facilitated by the consultant during Phase 2, the survey, available in English and Spanish, was developed to continue gathering community input and to validate what the consultant had heard in the community engagement meetings during Phase 1.
During Phase 1, the consultant facilitated twelve information gathering meetings with stakeholders, community members, staff, advisory board members, and representatives from local organizations and businesses. These meetings were designed to gather information on older adult needs and priorities in the City of Ventura.
City Council established a five member Senior Strategic Planning Ad Hoc Committee to support the development of the senior strategic plan and assist in engaging the community in the process. The Committee meets on the third Tuesday of each month from 1:30 - 2:30 pm in the Santa Cruz Meeting Room at City Hall. These meetings are open to the public with community members who are interested in sharing additional input encouraged to attend.
Committee Members: