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Methods and products for modulating an immune response are provided. Pharmaceutical preparations contain a conjugate of an antigen and a FcRn binding partner. The conjugates are administered to mammals in effective amounts to modulate the immune system by stimulating the immune response against the antigen or tolerizing the immune system to the antigen. The antigen may be characteristic of a pathogen, of an autoimmune disease or of an allergen.
1. A method for activating or suppressing a systemic immune response in a mammal comprising:
administering to the luminal side of an epithelial barrier of an epithelial tissue expressing FcRn receptor an effective amount of a conjugate of an FcRn binding partner and an antigen selected from group consisting of:
an antigen characteristic of a pathogen,
an antigen characteristic of an autoimmune disease, and
an antigen characteristic of an allergen.
2. The method of claim 1 wherein the conjugate is administered orally to the intestinal epithelium.
3. The method of claim 1 wherein the conjugate is administered in an aerosol to the lungs.
4. The method of claim 1 wherein the FcRn binding partner is a non-specific IgG or a FcRn binding fragment of IgG.
5. The method of claim 1 wherein the FcRn binding partner is an Fc fragment of IgG.
6. The method of any one of claims 1, 2, 3, 4, and 5, wherein the antigen is covalently coupled to the FcRn binding partner.
7. The method of any one of claims 1, 2, 3, 4, and 5, wherein the antigen is characteristic of an pathogen and wherein the conjugate is administered in an amount effective for activating an immune response.
8. The method of claim 7 wherein the antigen is covalently coupled to the FcRn binding partner.
9. The method of any one of claims 1, 2, 3, 4, and 5, wherein the antigen is selected from the group consisting of:
an antigen that is characteristic of an autoimmune disease, and
an antigen that is characteristic of an allergen, and wherein
the conjugate is administered in an amount effective to suppress the immune response.
10. The method of claim 1, wherein the mammal is a human.
11. The method of claim 10, wherein the human is an adult.
12. The method of claim 10, wherein the human is a child.
The work described herein was supported, in part, by National Institutes of Health Grant Nos. NIH DK-44319, NIH HO-27691 and NIH DK-48106. The U.S. Government has certain rights to this invention.
This invention relates in general to methods and products for initiating an immune response against an antigen, and in particular relates to transepithelial delivery of antigens to provoke tolerance and immunity.
The immune system of a mammal develops during gestation and becomes active in the late mammalian fetus. Although active, it still might be characterized as `immature`0 because it has not been challenged to any significant extent by antigens; the fetus is largely protected from antigens by the mother. This `immature` immune system, however, is supplemented by the transfer of material immunoglobulin to the fetus (or in some cases to the neonate) to provide humoral immunity during the first weeks of independent life.
Rats and mice receive most maternal immunoglobulin G (IgG) as sucklings from colostrum and milk, although some is acquired prenatally. Cattle also receive IgG from colostrum. In rabbits, IgG is transported to the fetus across the yolk sac. Little is known about the transfer of IgG to the fetus or neonate in humans. Most evidence suggests that human mothers transfer humoral immunity to an offspring only before birth, although IgA transferred to a neonate via breast milk is believed to play a role in protecting the neonate against enteric infection.
The delivery of maternal IgG to the mammalian and/or neonate requires transport across an epithelial barrier which is largely impervious to macromolecules. The transport of macromolecules across such an epithelial barrier may occur by non-specific and specific receptor-mediated mechanisms. Receptor non-specific mechanisms are represented by paracellular sieving events, the efficiency of which are inversely related to the molecular weight of the transported molecule. Transport of macromolecules such as IgG across this paracellular pathway is highly inefficient. Descriptions of receptor-mediated transport of immunoglobulins through intestinal epithelial cells are limited thus far to the polymeric immunoglobulin receptor and the enterocyte receptor for IgG (a major histocompatibility complex (MHC) class I related Fc receptor). These two receptor systems differ in their specificity for immunoglobulin isotype, in their direction of immunoglobulin transport across the epithelial cell and in their tissue-specific expression. Both may play a role in molding the immature immune system.
The polymeric immunoglobulin receptor is expressed on the basolateral surfaces of enterocytes, hepatocytes and/or biliary duct epithelial cells. It transports polymeric IgA and IgM to the apical (luminal) surfaces, concentrating these immunoglobulins for antimicrobial defense and antigen exclusion.
The enterocyte receptor for IgG, which has homology to the MHC class I heavy chain and is associated with beta2 -microglobulin (β2 M), is expressed on neonatal enterocytes of the rat and mouse. IgG is transported transcellularly in a luminal to serosal direction across the intestinal epithelium of these rodent neonates. On the apical surface of the enterocyte, the Fc portion of IgG is bound to the enterocyte receptor at the relatively acidic pH of the lumen (about pH 6.0). Following transcytosis to the basolateral plasma membrane, discharge of the immunoglobulin occurs at the relatively neutral pH of the interstitial fluids (about pH 7.4). The rodent neonatal Fc receptor (FcRn) therefore could be responsible for delivery of maternal IgG to the neonate and as such may be responsible for the passive acquisition of IgG during this period.
In humans, maternal IgG is actively transported across the placenta. The receptor responsible for this transport has been sought for many years. Several IgG-binding proteins have been isolated from placenta. FcγRII was detected in placental endothelium and FcγRIII in syncytiotrophoblasts. Both of these receptors, however, showed a relatively low affinity for monomeric IgG. Recently, the isolation from placenta of a cDNA encoding a human homolog of the rat and mouse enterocyte receptor for IgG was reported. (Story, C. M. et al., J. Exp. Med., Vol. 180:2377-2381, December 1994) The complete nucleotide and deduced amino acid sequence is reported. This Fc receptor for IgG may be responsible for the transport of maternal IgG to the human fetus (and even possibly to the neonate), as the molecule is highly homologous over its open reading frame with the rat FcRn sequence (69% nucleotide identity and 65% predicted amino acid identity). So called passive immunization in the human fetus (and possibly in the human neonate) now may become better understood.
In contrast to passive immunization which involves supplementing a host's immune system with antibodies derived from another, active immunization involves stimulation of the host's own immune system to generate in vivo the desired immune response. The most widely practiced methods of active immunization in children and adults involve injections of an immunogen, once as an initial dose and then at least once again as a booster dose. These methods suffer many serious drawbacks, including the risks associated with the use of needles that can transmit diseases such as AIDS and hepatitis. (When tolerizing a patient against an allergen, the problems are compounded in that repeated injections over a long period of time often are required.) These methods also do not necessarily trigger adequately the first line of defense against many pathogens, that is, mucosal immunity. Mucous membranes line the airways, the reproductive system and the gastrointestinal tract, and this mucosal surface represents the first portal of entry for many diseases. An oral vaccine that is easy to deliver and that triggers mucosal immunity would be highly desirable.
Immunization using oral vaccines is problematic. Often little or no immune response is achieved. To enhance the immune response, antigens of interest have been coupled to carriers that are known to be strongly immunogenic. For example, researchers have delivered antigens using Bacille Calmette-Gurein (BCG) as a carrier; BCG is a bacterium originally used as an oral vaccine against tuberculosis. A problem with such carriers is that the patient will develop antibodies against the carrier itself, which can be troublesome if the carrier is used again for delivering a different antigen to the same patient. To date, no general strategy for oral vaccines has proven successful.
Immunoglobulin and portions thereof in the past have been conjugated to drugs and imaging agents to target and destroy cell populations and to extend the half-lives of certain agents. Immunotoxins are an example of such conjugates. Such conjugates, however, have never been proposed as useful for initiating an immune response.
A small body of work has focused on the tolerogenic capacity of immunoglobulins coupled to oligonucleotides or proteins characteristic of autoimmune diseases. (See PCT WO 91/08773). This work is based upon the notion that the induction of tolerance may be strongly influenced by carrier moieties and that immunoglobulin carriers appear to be strongly tolerogenic. Isologous IgG is the preferred carrier, and intravenous administration was the mode used for delivering the conjugates of IgG. Although this body of work extends for more than a decade, oral administration is mentioned only once and only for conjugates where IgA is the immunoglobulin carrier. Thus, although tolerogenic immunoglobulin conjugates are known in the art, such conjugates have never been suggested as agents for inducing a robust response against an antigen characteristic of a pathogen. (To the contrary, the art suggests that such conjugates, if anything, would tolerize a subject against a pathogen which would be highly undesirable). In addition, it never has been suggested that such conjugates would be effective tolerogens when the immunoglobulin is IgG and the mode of delivery is oral delivery.
The invention involves the discovery that antigens may be coupled to molecules that bind to the FcRn receptor, such as immunoglobulins, or portions thereof, and delivered across epithelial barriers by active transport through the enterocyte via FcRn receptors. The immunoglobulin or portion thereof binds to the FcRn receptor and acts as a carrier for the antigen as the immunoglobulin or portion thereof is transported across the epithelial barrier by FcRn mediated transport. The FcRn receptor is present in the human epithelial tissue of children and adults, and the invention therefore permits effective strategies for immunizing humans.
According to one aspect of the invention, a method for modulating the immune system of a mammal is provided. An effective amount of a conjugate of an antigen and a FcRn binding partner is administered to an epithelial barrier of a mammal in need of such immune modulation. The antigen is selected from the group consisting of: an antigen that is characteristic of a pathogen, an antigen that is characteristic of an autoimmune disease, and an antigen that is characteristic of an allergen. In preferred embodiments, the FcRn binding partner is non-specific IgG or a FcRn binding fragment of IgG. Most preferably the FcRn binding partner is an Fc fragment of IgG. It also is preferred that the antigen be covalently coupled to the FcRn binding partner. Preferably the conjugate is administered orally to the intestinal epithelium, in an aerosol to the lungs or intranasally. Such preparations may be nonaseptic. Supplementary potentiating agents, as described below, may be administered in addition.
According to another aspect of the invention, a pharmaceutical preparation is provided. The pharmaceutical preparation includes a conjugate of an antigen and a FcRn binding partner, wherein the antigen is selected from the group consisting of: an antigen that is characteristic of a pathogen, an antigen that is characteristic of an autoimmune disease, and an antigen that is characteristic of an allergen. The preferred FcRn binding partners are as described above. The conjugate is present in an amount effective for modulating the immune response of a mammal. The pharmaceutical preparation also includes a pharmaceutically acceptable carrier. When the antigen is characteristic of an autoimmune disease or an allergen, then the pharmaceutical preparations of the invention must be formulated in unit dosage form constructed and arranged for delivery to an epithelial carrier such as for oral delivery to the intestinal epithelium, aerosol delivery to the pulmonary epithelium and intranasal delivery to the nasal epithelium. Thus tablets containing IgG (or an FcRn binding portion thereof) coupled to any of the antigens as characterized above are embraced by the present invention.
The foregoing pharmaceutical preparations may be delivered together with supplementary potentiating agents including adjuvants, cytokines, bioadhesives and the like. The supplementary potentiating agents themselves may be coupled to a FcRn binding partner to facilitate the delivery of such agents across the epithelial barrier. Preferred modes of administration in general include oral dosages to the intestinal epithelium, aerosols to the lungs and intranasal dosages.
According to still another aspect of the invention, a method for making an immunomodulator is provided. The method involves covalently coupling an antigen or a supplementary potentiating agent to an FcRn binding partner, wherein the antigen or supplementary potentiating agent is selected as described above. The preferred FcRn binding partner also is as described above. The conjugates then can be used to prepare the pharmaceutical preparations for modulating a mammal's immune response as described above.
In yet another aspect of the invention, the conjugate including the antigen crosses the epithelial barrier in an amount at least double the extent that the antigen crosses the epithelial barrier in an unconjugated form. It thus is an object of the invention to develop a mechanism for increasing the ability of an antigen to cross an epithelial barrier.
Another object of the invention is to develop a new class of orally active immunogens and toleragens.
Another object of the invention is to develop improved methods for stimulating mucosal immunity.
These and other aspects of the invention are described in greater detail below.
The invention involves the discovery that the human FcRn receptor is active in adult epithelial tissue and the discovery that FcRn binding partners such as IgG or Fc fragments can be used to transport other molecules, including antigens, across epithelial barriers. In this manner, FcRn binding partners such as IgG or an FcRn binding portion thereof can be used to deliver an antigen across an epithelial barrier to a subject's immune system, thereby initiating an immune response.
The invention is useful whenever it is desirable to enhance the delivery of an antigen across an epithelial barrier to the immune system. The invention thus may be used to deliver antigens across intestinal epithelial tissue, lung epithelial tissue and other mucosal surfaces including nasal surfaces, vaginal surfaces, colon surfaces and biliary tree surfaces. The invention may be used to modulate a subject's immune system such as by stimulating a humoral antibody response against an antigen, by stimulating T cell activity, or by stimulating tolerance to an antigen. As used herein, subject means: humans, primates, horses, cows, sheep, pigs, goats, dogs, cats, chickens and rodents.
The invention involves the formation of a conjugate of an FcRn binding partner and an antigen. By conjugate it is meant two entities bound to one another by any physiochemical means, including hydrophobic interaction between an antigen and the non-specific hydrophobic portions of an antibody molecule, antibody-antigen specific binding and covalent coupling. The nature of the preferred bonding will depend, among other things, upon the mode of administration and the pharmaceutical carriers used to deliver the conjugate to the selected epithelial barrier. For example, some bonds are not as well suited as others to withstand certain environments such as the stomach, but can be protected therefrom by delivery systems which bypass the stomach. It, of course, is important that the bond between the FcRn binding partner and the antigen be of such a nature that it does not destroy the ability of the FcRn binding partner to bind to the FcRn receptor. Such bonds are well known to those of ordinary skill in the art; examples are provided in greater detail below. The conjugate further may be formed as a fusion protein, also discussed in greater detail below.
An FcRn binding partner means any entity that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. As mentioned above, the FcRn receptor has been isolated for several mammalian species, including humans. The sequence of the human FcRn, rat FcRn and mouse FcRn may be found in Story, C. M. et al, J. Exp. Med., vol. 180:2377-2381, December 1994. The FcRn receptor molecule now is well characterized. The FcRn receptor binds IgG (but not other immunoglobulin classes such as IgA, IgD, IgM and IgE) at a relatively lower pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at a relatively higher pH found in the interstitial fluids. As will be recognized by those of ordinary skill in the art, FcRn receptors can be isolated by cloning or by affinity purification using, for example, monoclonal antibodies. Such isolated FcRn receptors then can be used to identify and isolate FcRn binding partners, as described below.
FcRn binding partners include whole IgG, the Fc fragment of IgG and other fragments of IgG that include the complete binding region for the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based upon X-ray crystallography (Burmeister, W. P. et al., Nature, 1994; 372:379-378.) The major contact area of Fc with the FcRn receptor is near the junction of the CH 2 and CH 3 domains. Potential contacts are residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 in CH 2 and 385-387, 428 and 433-436 in CH 3. (These sites are distinct from those identified by subclass comparison or by site-directed mutagenesis as important for Fc binding to leukocyte FcγRI and FcγRII.) The foregoing Fc-FcRn contacts are all within a single Ig heavy chain. It has been noted previously that two FcRn receptors can bind a single Fc molecule. The crystallographic data suggest that in such a complex, each FcRn molecule binds a single polypeptide of the Fc homodimer.
Given the foregoing information, those of ordinary skill in the art will readily recognize that the Fc region of IgG can be modified according to well-recognized procedures such as site-directed mutagenesis and the like to yield modified IgG or modified Fc fragments or portions thereof that will be bound by the FcRn receptor. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding. In addition, other binding partners can be identified and isolated. Antibodies or portions thereof specific for the FcRn receptor and capable of being transported by FcRn once bound can be identified and isolated using well established techniques. Likewise, random generated molecularly diverse libraries can be screened and molecules that are bound and transported by FcRn receptors can be isolated using conventional techniques. It is not intended that the invention be limited by the selection of any particular FcRn binding partner. Where the binding partner is IgG or a FcRn binding portion thereof, the IgG or portion thereof may be prepared according to conventional procedures as described in greater detail below.
The FcRn binding partner is conjugated with an antigen. An antigen as used herein falls into three classes: 1) antigens that are characteristic of a pathogen; 2) antigens that are characteristic of an autoimmune disease; and 3) antigens that are characteristic of an allergen. Antigens in general include polysaccharides, glycolipids, glycoproteins, peptides, proteins, carbohydrates and lipids from cell surfaces, cytoplasm, nuclei, mitochondria and the like.
Antigens that are characteristic of pathogens include antigens derived from viruses, bacteria, parasites or fungi. Examples of important pathogens include vibrio cholerae, enterotoxigenic escherichia coli, rotavirus, clostridium difficile, shigella species, salmonella typhi, parainfluenza virus, influenza virus, streptococcus pneumoniae, borella burgdorferi, HIV, streptococcus mutans, plasmodium falciparum, staphylococcus aureus, rabies virus and Epstein-Barr virus.
Viruses in general include but are not limited to those in the following families: picornaviridae; caliciviridae; togaviridae; flaviviridae; coronaviridae; rhabdoviridae; filoviridae; paramyxoviridae; orthomyxoviridae; bunyaviridae; arenaviridae; reoviridae; retroviridae; hepadnaviridae; parvoviridae; papovaviridae; adenoviridae; herpesviridae; and poxyviridae.
Bacteria in general include but are not limited to: P. aeruginosa; E. coli; Klebsiella sp.; Serratia sp.; Pseudomonas sp.; P. cepacia; Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumoniae; S. aureus; Haemophilus sp.; Neisseria sp.; N. meningitidis; Bacteroides sp.; Citrobacter sp.; Branhamella sp.; Salmonella sp.; Shigella sp.; S. pyogenes; Proteus sp.; Clostridium sp.; Erysipelothrix sp.; Lesteria sp.; Pasteurella multocida; Streptobacillus sp.; Spirillum sp.; Fusospirocheta sp.; Treponema pallidum; Borrelia sp.; Actinomycetes; Mycoplasma sp.; Chlamydia sp.; Rickettsia sp.; Spirochaeta; Legionella sp.; Mycobacteria sp.; Ureaplasma sp.; Streptomyces sp.; Trichomoras sp.; and P. mirabilis.
Parasites include but are not limited to: Plasmodium falciparum, P. vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana, L. tropica, L. major, L. aethiopica, L. donovani; Trypanosoma cruzi, T. brucei; Schistosoma mansoni, S. haematobium, S.japonium; Trichinella spiralis; Wuchereria bancrofti; Brugia malayi; Entamoeba histolytica; Enterobius vermiculoarus; Taenia solium, T. saginata; Trichomonas vaginatis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidium parvum; Pneumocytis carinii; Babesia bovis, B. divergens, B. microti; Isospora belli, I. hominis; Dientamoeba fragilis; Onchocerca volvulus; Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale; Strongyloides stercoralis; Capillaria philippinensis; Angiostrongylus cantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcus granulosus, E. multilocularis; Paragonimus westermani, P. caliensis; Chlonorchis sinensis; Opisthorchis felineus, O. viverrini; Fasciola hepatica; Sarcoptes scabiei; Pediculus humanus; Phthirius pubis; and Dermatobia hominis.
Fungi in general include but are not limited to: Cryptococcus neoformans; Blastomyces dermatitidis; Ajellomyces dermatitidis; Histoplasma capsulatum; Coccidioides immitis; Candida species, including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii and C. krusei; Aspergillus species, including A. fumigatus, A. flavus and A. niger; Rhizopus species; Rhizomucor species; Cunninghammella species; Apophysomyces species, including A. saksenaea, A. mucor and A. absidia; Sporothrix schenckii; Paracoccidioides brasiliensis; Pseudallescheria boydii; Torulopsis glabrata; and Dermatophytes species.
Antigens that are characteristic of autoimmune disease typically will be derived from the cell surface, cytoplasm, nucleus, mitochondria and the like of mammalian tissues. Examples include antigens characteristic of uveitis (e.g. Santigen), diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, primary myxoedema, thyrotoxicosis, rheumatoid arthritis, pernicious anemia, Addison's disease, scleroderma, autoimmune atrophic gastritis, premature menopause (few cases), male infertility (few cases), juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic opthalmia, phacogenic uveitis, autoimmune haemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leucopenia, primary biliary cirrhosis (few cases), ulcerative colitis, Sjogren's syndrome, Wegener's granulomatosis, poly/dermatomyositis, and discoid lupus erythromatosus.
Antigens that are allergens are generally proteins or glycoproteins, although allergens may also be low molecular weight allergenic haptens that induce allergy after covalently combining with a protein carrier (Remington's Pharmaceutical Sciences). Allergens include antigens derived from pollens, dust, molds, spores, dander, insects and foods. Specific examples include the urushiols (pentadecylcatechol or heptadecylcatechol) of Toxicodendron species such as poison ivy, poison oak and poison sumac, and the sesquiterpenoid lactones of ragweed and related plants.
In the cases of protein and peptide antigens, covalent linking to an FcRn partner is intended to include linkage by peptide bonds in a single polypeptide chain. Established methods (Sambrook, Ausubel) would be used to engineer DNA encoding a fusion protein comprised of the antigenic peptide or protein and an FcRn partner. This DNA would be placed in an expression vector and introduced into bacterial or eukaryotic cells by established methods. The fusion protein would be purified from the cells for from culture medium by established methods.
When administered, the conjugates of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines, and optionally other therapeutic agents. Thus, "cocktails" including the conjugates and the agents are contemplated. The agents themselves may be conjugated to FcRn binding partners to enhance delivery of the agents across the epithelial barriers.
The conjugates of the invention may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicyclic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceutically acceptable salts can be prepared as alkyline metal or alkyline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); sodium bicarbonate (0.5-1.0% W/V); and phosphoric acid and a salt (0.8-2% W/V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).
The term "carrier" as used herein, and described more fully below, means one or more solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other mammal. The "carrier" may be an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate administration.
The components of the pharmaceutical compositions are capable of being comingled with the conjugates of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. The components of oral drug formulations include diluents, binders, lubricants, glidants, disintegrants, coloring agents and flavoring agents. Encapsulating substances for the preparation of enteric-coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate and methacrylic acid ester copolymers. Solid oral formulations such as capsules or tablets are preferred. Elixirs and syrups also are well known oral formulations. The components of aerosol formulations include solubilized active ingredients, antioxidants, solvent blends and propellants for solution formulations, and micronized and suspended active ingredients, dispersing agents and propellants for suspension formulations. The oral, aerosol and nasal formulations of the invention can be distinguished from injectable preparations of the prior art because such formulations may be nonaseptic, whereas injectable preparations must be aseptic.
The term "adjuvant" is intended to include any substance which is incorporated into or administered simultaneously with the conjugates of the invention and which nonspecifically potentiates the immune response in the subject. Adjuvants include aluminum compounds, e.g., gels, aluminum hydroxide and aluminum phosphate, and Freund's complete or incomplete adjuvant (in which the conjugate is incorporated in the aqueous phase of a stabilized water in paraffin oil emulsion). The paraffin oil may be replaced with different types of oils, e.g., squalene or peanut oil. Other materials with adjuvant properties include BCG (attenuated Mycobacterium tuberculosis), calcium phosphate, levamisole, isoprinosine, polyanions (e.g., poly A:U) leutinan, pertussis toxin, cholera toxin, lipid A, saponins and peptides, e.g. muramyl dipeptide. Rare earth salts, e.g., lanthanum and cerium, may also be used as adjuvants. The amount of adjuvants depends on the subject and the particular conjugate used and can be readily determined by one skilled in the art without undue experimentation.
Other supplementary immune potentiating agents, such as cytokines, may be delivered in conjunction with the conjugates of the invention. The cytokines contemplated are those that will enhance the beneficial effects that result from administering the immunomodulators according to the invention. Cytokines are factors that support the growth and maturation of cells, including lymphocytes. It is believed that the addition of cytokines will augment cytokine activity stimulated in vivo by carrying out the methods of the invention. The preferred cytokines are interleukin (IL)-1, IL-2, gamma-interferon and tumor necrosis factor α. Other useful cytokines are believed to be IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, erythropoietin, leukemia inhibitory factor, oncostatin-M, ciliary neurotrophic factor, growth hormone, prolactin, CD40-ligand, CD27-ligand, CD30-ligand, alpha-interferon, beta-interferon, and tumor necrosis factor-β. Other cytokines known to modulate T-cell activity in a manner likely to be useful according to the invention are colony stimulating factors and growth factors including granulocyte and/or macrophage stimulating factors (GM-CSF, G-CSF and CSF-1) and platelet derived, epidermal, insulin-like, transforming and fibroblast growth factors. The selection of the particular cytokines will depend upon the particular modulation of the immune system that is desired. The activity of cytokines on particular cell types is known to those of ordinary skill in the art.
The precise amounts of the foregoing cytokines used in the invention will depend upon a variety of factors, including the conjugate selected, the dose and dose-timing selected, the mode of administration and the characteristics of the subject. The precise amounts selected can be determined without undue experimentation, particularly since a threshold amount will be any amount which will enhance the desired immune response. Thus, it is believed that nanogram to milligram amounts are useful, depending upon the mode of delivery, but that nanogram to microgram amounts are likely to be most useful because physiological levels of cytokines are correspondingly low.
The preparations of the invention are administered in effective amounts. An effective amount is that amount of a conjugate that will alone, or together with further doses, stimulate an immune response as desired. This may involve the stimulation of a humoral antibody response resulting in an increase in antibody titer in serum, improved mucosal immunity, a clonal expansion of cytotoxic T lymphocytes or tolerance to an antigen, including a self antigen. It is believed that doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration, will be effective. The preferred range is believed to be between about 500 nanograms and 500 micrograms/kilogram, and most preferably between 1 microgram and 100 micrograms/kilogram. The absolute amount will depend upon a variety of factors, including the conjugate selected, the immune modulation desired, whether the administration is in a single or multiple doses, and individual patient parameters including age, physical condition, size and weight. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular conjugate selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, involve delivering the conjugates of the invention to an epithelial surface. Preferred modes of administration are oral, intrapulmonary, intrabiliary and intranasal.
Compositions may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the conjugate into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the conjugate into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the conjugates of the invention, increasing convenience to the subject and the physician. Many types of release/delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhidrides and polycaprolactone; wax coatings, compressed tablets using conventional binders and excipients, and the like. Bioadhesive polymer systems to enhance delivery of a material to the intestinal epithelium are known and described in published PCT application WO 93/21906. Capsules for delivering agents to the intestinal epithelium also are described in published PCT application WO 93/19660.
BSA, bovine serum albumin; cDNA, complementary deoxyribonucleic acid; CT-B, cholera toxin B subunit; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; DOC, desoxycholate; ECL, enhanced chemiluminescence; ELISA, enzyme linked immunosorbant assay; HBSS, Hanks' balanced salt solution without calcium or magnesium; HEPES, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; hGH, human growth hormone; IEC, intestinal epithelial cells; KI, potassium iodide; MHC, major histocompatibility complex; NaOH, sodium hydroxide; NH4 Cl, ammonium chloride; NHS-rhodamine, N-hydroxysuccinimidyl-rhodamine; RNA, ribonucleic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; SATA, N-succinimidyl S-acetylthioacetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; sulfo-LC-SPDP, sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamide] hexanoate; sulfo-NHS-biotin, sulfosuccinimidobiotin; sulfo-SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclo-hexane-1-carboxylate.
cDNA Cycle Kits was purchased from Invitrogen (San Diego, Calif.). Taq polymerase was purchased from Perkin-Elmer Cetus (Norwalk, Conn.). CircumVent™ Kits were purchased from New England Biolabs (Beverly, Mass.). Radionuclides and radioactive chemicals were purchased from DuPont/NEN (Boston, Mass.). HBSS- and DMEM were purchased from GIBCO/Life Technologies (Gaithersburg, Md.). RPMI 1640 was purchased from Cellgro (Herndon, Va.). L-glutamine was purchased from Cellgro. Protein A-Sepharose was purchased from Pharmacia Biotech (Piscataway, N.J.). Streptavidin-horseradish peroxidase, sulfo-LC-SPDP, sulfo-NHS-biotin, sulfo-SMCC, SATA and immobilized ficin were purchased from Pierce (Rockford, Ill.). Balb/c mice were purchased from Charles River Laboratories (Wilmington, Mass.). ECL kits were purchased from Amersham (Arlington Heights, Ill.). Plasmin, AvidChrom-protein A, protein G-Sepharose, BSA, cholera toxin B subunit, anti-hGH antibodies and all other chemicals were purchased from Sigma (St. Louis, Mo.).
Expression of FcRn mRNA in Human Intestinal Epithelial Primary Cells and Cell Lines
Total RNA was extracted from adult human enterocytes by standard methodology well known in the art (Sambrook et al., ibid.). One microgram of RNA from each cell type was used as a template to prepare the cDNA substrate for reverse transcriptase-polymerase chain reaction (RT-PCR) using a cDNA Cycle Kit (Invitrogen, San Diego, Calif.). Thirty cycles of PCR were performed on the cDNA using Taq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) according to the manufacturer's instructions using primers TGCTGGGCTGTGAACTG (SEQ ID NO:1 and CGCTTTTAGCAGTCGGAA (SEQ ID NO:2. The PCR cycle conditions were: denaturation at 94� C. for one minute, annealing at 55� C. for two minutes and extension at 72� C. for three minutes. Amplification products were resolved by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining, which showed the presence of the expected approximately 800 base pair amplification product in all samples except the adult colonic epithelial cells. To confirm the identity of the RT-PCR amplification product, the DNA band was excised from the agarose gel, subcloned into pCR II (Invitrogen, San Diego, Calif.) and sequenced using a Prism N dye-deoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif.) using primers from both vector and human FcRn sequence. Reaction products were analyzed on an Applied Biosystems sequencer. The sequence of the amplification products exactly matched the FcRn gene sequence, confirming the identity of the expressed gene.
Detection of FcRn mRNA by Northern Blot
To confirm the expression of FcRn in human intestinal epithelial cells and cell lines, a Northern blot was prepared using the RNA samples prepared as described in Example 1 from adult human enterocytes, and from two human adenocarcinoma cell lines of colonic origin, CaCO-2 and HT-29. The RNA samples were resolved by formaldehyde/agarose gel electrophoresis and transferred to a nylon membrane by standard procedures (Sambrook or Ausubel). The membrane was probed using a 32 P-radiolabeled 120 base pair probe from the 3' untranslated region of FcRn by standard methods. Autoradiograms of the Northern blot demonstrated the presence of the 1.5 kilobase hFcRn transcript in the enterocytes and both cell lines. Therefore, the expression of FcRn in human adult intestinal epithelial cells and cell lines was demonstrated by two different methods of RNA detection.
Labeling and Immunoprecipitation of the MHC-Class I Related Fc Receptor (FcRn) from Intestinal Epithelial Cells
The expression of FcRn in human intestinal epithelial cells was confirmed by immunoprecipitation of the protein. Caco-2 cells were labeled metabolically using 35 S-methionine (DuPont/NEN, Boston, Mass.) and proteins were extracted by methods well known in the art (Harlow and Lane, Antibodies: A Laboratory Manual). A polyclonal rabbit anti-rat MHC class I related FcR heavy chain specific antiserum bound to protein-A-sephorose was used to immunoprecipitate FcRn from the cell extracts using standard methods (FcRn can be purified by well established methods, Simister and Rees 1985, European J. Immunology, 15:733-8, and used to immunize rats followed by collection of serum, Harlow and Lane, supra.). Immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography. A 48 kilodalton FcRn protein was observed, confirming expression observed at the RNA level.
Expression of FcRn Protein on the Cell Surface of Human Intestinal Epithelial Cells
About 3�107 HT-29 intestinal epithelial cells were detached from tissue culture plates by nonenzymatic methods and were washed four times with ice cold Hanks' balanced salt solution containing no calcium or magnesium (HBSS-,GIBCO/Life Technologies, Gaithersburg, Md.). To label cell surface proteins, the washed cells were incubated twice for 20 minutes with 1.5 ml of 0.5 mg/ml sulfo-NHS-biotin (Pierce, Rockford, Ill.) in DMSO. Labeled cells were washed five times with 50 mM NH4 Cl, incubated 20 minutes with 10 ml of RPMI 1640 (Cellgro, City, State) containing 1 mM L-glutamine (Mediatech, Washington, D.C.), and washed four times with HBSS-. The cells were lysed, then precleared overnight with protein A-Sepharose beads (Pharmacia Biotech, Piscataway, N.J.) using standard techniques well known in the art. SDS and desoxycholic acid (DOC) were added to the supernatants to final concentrations of 0.1% and 0.5%, respectively. Lysates were precleared with normal rabbit serum and immunoprecipitated with polyclonal rabbit anti-rat MHC class I related FcR antibody by methods well known in the art. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The nitrocellulose membrane was processed for incubation with 1:10,000 diluted streptavidin-horseradish peroxidase (Pierce, Rockford Ill.) as recommended by the manufacturer. The membrane was then processed for detection of bound horseradish peroxidase using an ECL kit (Amersham, Arlington Heights, Ill.). Light emitted by cleavage of the chemiluminescent substrate was detected by exposure of the membrane to light-sensitive film. The film exposures showed that FcRn was expressed on the surface of HT-29 intestinal epithelial cells.
Functional Activity of Human FcRn on the Cell Surface of Intestinal Epithelial Cells
To show that the FcRn expressed on the cell surface of intestinal epithelial cells was functional, Caco-2 cells and human adult jejunal intestinal epithelial cells (IECs) were tested for the ability to bind Fc fragment of antibody. Caco-2 and jejunal IECs were distributed to microcentrifuge tubes (2�106 cells per tube) and pelleted at 2000 rpm for 2-3 minutes at 4� C. Cell pellets were washed once in DMEM containing 20 mM HEPES, pH 6.0 or pH 8.0 at 4� C. and resuspended in 0.2 ml of the same medium. The cell suspensions were transferred to 12 well plates for assay.
125 I-Fc fragment (200 ng/ml, 4�10-9 M) in DMEM containing 20 mM HEPES, 1.0 mM KI and 0.1% fish gelatin, pH 6.0 or pH 8.0 with or without 0.5 mg/ml unlabeled human IgG (3.3�10-6 M) was added to each well. The cells were allowed to bind IgG or Fc at 37� C. for two hours in a 5% CO2 humidified atmosphere. Cells were transferred to microcentrifuge tubes and pelleted at 2000 rpm for 2-3 minutes at 4� C. Unbound 125I-Fc was removed by washing the cell pellets once with cold DMEM containing 20 mM HEPES, pH 6.0 or pH 8.0 at 4� C. Cells were disrupted in 0.5 ml 0.1 M NaOH and the resulting solution transferred to scintillation vials. 125 I was quantified using a CliniGamma 1272 gamma counter (LKB Wallac, Piscataway, N.J.). Both Caco-2 cells and human adult jejunum IECs specifically bound 125 I-Fc at pH 6.0 but not at pH 8.0, demonstrating functional pH-dependent binding as observed for rat neonatal FcRn and cloned human FcRn (Story et al., J. Exp. Med. 180:2377-2381; December 1994).
Preparation of Human Immunoglobulin G
Non-specific purified immunoglobulin G from human, mouse, rat, goat, pig, cow, and other species may be purchased from commercial vendors such as Sigma Chemical Co., Pierce Chemical, HyClone Laboratories, ICN Biomedicals and Organon Teknika-Cappel.
Immunoglobulin G also may be isolated by ammonium sulfate precipitation of precipitation of blood serum. The protein precipitate is further fractionated by ion exchange chromatography or gel filtration chromatography, to isolate substantially purified non-specific IgG. By non-specific IgG it is meant that no single specificity within the antibody population or pool is dominant.
Immunoglobulin G also may be purified from blood serum by adsorption to protein A attached to a solid support such as protein A-Sepharose (Pharmacia), AvidChrom-Protein A (Sigma), or protein G-Sepharose (Sigma). Other methods of purification of IgG are well known to persons skilled in the art and may be used for the purpose of isolation of non-specific IgG.
Preparation of Human Immunoglobulin G Fc Fragment
To prepare the Fc fragments of human IgG, IgG isolated as in example 6 is subjected to digestion with immobilized papain (Pierce) according to the manufacturer's recommended protocol. Other proteases that digest IgG to produce intact Fc fragments that can bind to Fc receptors, e.g. plasmin (Sigma) or immobilized ficin (Pierce), are known to skilled artisans and may be used to prepare Fc fragments. The digested immunoglobulin then is incubated with an affinity matrix such as protein A-Sepharose or protein G-Sepharose. Non-binding portions of IgG are eluted from the affinity matrix by extensive washing in batch or column format. Fc fragments of IgG then are eluted by addition of a buffer that is incompatible with Fc-adsorbant binding. Other methodologies effective in the purification of Fc fragments also may be employed.
Conjugation of Compounds to Human Immunoglobulin Fc Fragments
To deliver compounds via the FcRn transport mechanism, such compounds can be coupled to whole IgG or Fc fragments. The chemistry of cross-linking and effective reagents for such purposes are well known in the art. The nature of the crosslinking reagent used to conjugate whole IgG or Fc fragments and the compound to be delivered is not restricted by the invention. Any crosslinking agent may be used provided that a) the activity of the compound is retained, and b) binding by the FcRn of the Fc portion of the conjugate is not adversely affected.
An example of an effective one-step crosslinking of Fc and a compound is oxidation of Fc with sodium periodate in sodium phosphate buffer for 30 minutes at room temperature, followed by overnight incubation at 4� C. with the compound to be conjugated. Conjugation also may be performed by derivatizing both the compound and Fc fragments with sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamide]hexanoate (sulfo-LC-SPDP, Pierce) for 18 hours at room temperature. Conjugates also may be prepared by derivatizing Fc fragments and the desired compound to be delivered with different crosslinking reagents that will subsequently form a covalent linkage. An example of this reaction is derivatization of Fc fragments with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclo-hexane-1-carboxylate (Sulfo-SMCC, Pierce) and the compound to be conjugated to Fc is thiolated with N-succinimidyl S-acetylthioacetate (SATA). The derivatized components are purified free of crosslinker and combined at room temperature for one hour to allow crosslinking. Other crosslinking reagents comprising aldehyde, imide, cyano, halogen, carboxyl, activated carboxyl, anhydride and maleimide functional groups are known to persons of ordinary skill in the art and also may be used for conjugation of compounds to Fc fragments. In all of the above crosslinking reactions it is important to purify the derivatized compounds free of crosslinking reagent. It is important also to purify the final conjugate substantially free of unconjugated reactants. Purification may be achieved by affinity, gel filtration or ion exchange chromatography based on the properties of either component of the conjugate. A particularly preferred method is an initial affinity purification step using protein A-Sepharose to retain Fc and Fc-compound conjugates, followed by gel filtration or ion exchange chromatography based on the mass, size or charge of the Fc conjugate. The initial step of this purification scheme ensures that the conjugate will bind to FcRn which is an essential requirement of the invention.
IgG-Facilitated Delivery of Foreign Antigen Across the Intestinal Epithelial Barrier
To test the ability of Fc binding partner-antigen conjugates to be transported across epithelial barriers, foreign antigens are conjugated to IgG molecules for administration to mice. A convenient foreign antigen is the fluorescent dye rhodamine, since it may be visualized in frozen semi-thin sections of intestinal epithelium. Rhodamine is covalently linked to non-specific mouse IgG, prepared as described in Example 6, cholera toxin B subunit (Sigma) and ovalbumin (Sigma) by incubation with succinyl-rhodamine (Molecular Probes, Eugene, Oreg.) as recommended by the manufacturer. The IgG-rhodamine conjugate is purified by protein G-Sepharose affinity chromatography. After dialysis to remove unconjugated succinyl-rhodamine, cholera toxin B (CT-B)-rhodamine and ovalbumin-rhodamine conjugates are purified by gel filtrations or ion exchange chromatography. Fractions of the eluate are assayed for the presence of conjugates by determining fluorescence. Functional binding of the IgG and CT-B subunit conjugates may be tested by binding to FcRn and ganglioside GMl, respectively. Cholera toxin B-rhodamine and ovalbumin-rhodamine serve as positive and negative controls, respectively.
Balb/c mice are administered 0.2 nanomoles of the three rhodamine cojugates described above, with or without 0.2 nanomoles unlabeled cholera toxin as a non-specific adjuvant, by intragastric administration in the presence of 75 micromoles NaHCO3 and 20 mg/ml soybean trypsin inhibitor to inhibit gastric degradation. After 6 hours the mice are sacrificed and intestine is removed, frozen and processed for semi-thin sectioning. Sections of the intestinal epithelium are illuminated on a fluorescent microscope and examined for intracellular fluorescence. The presence of fluorescence in intestinal epithelial cells of mice fed IgG-rhodamine indicates that the IgG conjugates are effectively transported in an apical to basolateral direction across the intestinal epithelial barrier. FcRn is capable of transporting immunogens as conjugates with FcRn binding partners.
Mouse Mucosal Immune Response to Orally Delivered Antigen-IgG Conjugate Via FcRn-Mediated Transcytosis
Transgenic mice homozygous for deletion of β2 -microglobulin (a critical component of Fc-receptor function) and their normal wild-type litter mates are used for studies of generation of a mucosal immune response. If rhodamine-IgG elicits a mucosal immune response by binding to apical membrane Fc receptors, a positive immune response should be found in wild-type but not β2 -microglobulin "knockout" mice. In contrast, rhodamine-cholera toxin B subunit (CT-B) should elicit a positive immune response in both wild type and "knockout" mice as transcytosis of CT-B across the epithelial barrier is not dependent on binding to apical membrane Fc receptors. Rhodamine-ovalbumin does not enter transcytotic vesicles (but may enter intestinal epithelia by fluid phase endocytosis) and should not elicit an immune response in any mice.
Three groups of wild type and β2 -microglobulin knockout mice are orally immunized with the three rhodamine conjugates described in Example 9. Parallel experiments are conducted with the addition of 0.2 nanomoles of cholera toxin as non-specific adjuvant. Equimolar quantities of the rhodamine conjugates are administered intragastrically. The mice are "immunized" by this method every ten days for a total of three times. Two weeks after the third oral immunization the mice are sacrificed and the rhodamine-specific immune response is determined by ELISA on gut secretions and serum by standard methodology. Anti-rhodamine serum immunoglobulins are most evident in the wild type mice fed rhodamine conjugates of CT-B and IgG. Knockout mice lacking β2 -microglobulin generate a mucosal immune response to rhodamine-CT-B but not to rhodamine-IgG, indicating that receptor-mediated transcytosis plays an essential role in the mucosal immune response. The control rhodamine-ovalbumin conjugate elicits little or no immune response in either the wild type or the β2 -microglobulin knockout mice.
Those skilled in the art will be able to recognize or ascertain with no more than routine experimentation numerous equivalents to the specific products and processes described above. Such equivalents are considered to be within the scope of the invention and are intended to be covered by the following claims.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 2- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 17 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:# 17 TG- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:# 18 GAA__________________________________________________________________________
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Most trade speculation in the National Football League is hilarious, because it looks like a trade you'd make in a fantasy football league. Anyone remember the Justin Hunter for Isaiah Crowell rumor before the Titans/Browns game? Yeah, they are kind ...Read More »
Podcasts of the Week
For many commuters, the podcast has replaced the radio as the go-to source for entertainment. I'd say more specifically, in my case the podcast replaced the sports radio station a few years ago to the point that I barely remember ...Read More »
10 Fantasy Football Forums Worth Checking Out
In the summer of 2010 I started writing. First, it was writing about the Tampa Bay Rays — my favorite baseball team — on my blog (which is no longer updated). Eventually, when football rolled around, I was writing about ...Read More »
6 Best Fantasy Football Draft Apps For iPads in 2013
While Fantasy Football draft apps certainly make your draft easier, you still have to get the right ones and know how to use it. Have you ever noticed how the easier life becomes, other things become more difficult? For instance, ...Read More »
FS Interview Series: Meet Sigmund Bloom
Meet Sigmund Bloom, lover of fantasy football and…Phish? I first heard Sigmund on Nashville's Wake Up Zone morning radio show. He was answering WDIS (Who Do I Start?) calls and e-mails on the air at a furious pace. Well, the radio ...Read More »

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Richard Graham
Richard Graham, a Conservative British lawmaker, has suggested that young women who wear high heels and short skirts put themselves at greater risk of rape has drawn widespread condemnation.
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"The holiday cards were phenomenal ! Design services were beyond professional and streamlined the design to perfection. I just adore these cards!!!"
"This card met all my expectations. This year I was set on getting a card that would reflect a modern twist to christmas! I love the foil and the options to choose what color foil would go with your image. I'm a veteran to tiny prints and always can depend on there quality, and design."
"We used a selfie for our photo this year and the quality of the resolution turned out fine for the size of this card. We were also able to add copy to the back of the card---a nice addition!"
"I received my cards today (Monday, Dec. 15th) a day sooner than expected. I did not have time to take advantage of the review of the product prior to shipping, but I am very pleased with the picture quality, color and the weight of the paper, quality of the printing and the letterpress. Definitely the contemporary look I was after."


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Impax Asian Environmental Markets plc
Issue of equity
The Company announces that, following an exercise of subscription shares, it has issued 4,515 ordinary shares in the Company for cash at a price of 100p^ per share.
Following the issue of these shares, the Company's share capital consists of 214,990,197 ordinary shares of 1p each, carrying one voting right each and 39,000,394 subscription shares of 0.1p each, carrying no voting rights. The Company holds no shares in Treasury.
Therefore, the total number of voting rights for ordinary shareholders is 214,990,197. For shareholders holding both ordinary shares and subscription shares, the total number of voting rights is 253,990,591.*
The above figures may be used by shareholders as denominators for the calculations by which they will determine if they are required to notify their interest in, or a change of interest in, the Company under the FSA's Disclosure and Transparency Rules.
^ Subscription shareholders have the right, on any business day up to and including 31 October 2014, to subscribe for all or any of the ordinary shares to which their subscription shares relate at a price of 100 pence per ordinary share.
* The subscription shares are considered to be qualifying financial instruments under the FSA's Disclosure and Transparency Rules.
For further information please contact:
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7 July 2011
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The first visible minority national talk show host in Canada turns her back on the progressive faith.
Rejecting the Left
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The Limits of Power
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One of the best buys in my opinion. Can't wait until I get the hand one. It can get hard to push because essentially you're pushing a towel but I'm also a wimp. Works really well though. I've never had it clog. It does say do not add anything BUT water to it. I have sprayed the floors with a cleaner before and still no problems.
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havn't seen one so id like to to start it.
ok heres my Car
havn't seen one so id like to to start it.
ok heres my Car
best of both worlds
hears mine
Nothing Special. No bags. No coils. Stock except, some springs, a flash upgrade and an SRI.
Northshore Auto Union(N.S.A.U.)-Salem, Ma.
MKV Rabbit aka The Panda
pre wreck
BooBoo2KGolf // Ben // IG: LivinLow401
Originally Posted by SlayStation:
my toy... coming out again, soon
Almost done!
"Your pants too tight,your wheels too bright"
**** diabetes!
That brown GTI is awesome!
My little 2.slow...
Rhode worthy this year. Took her for a spin today
Guru Motorsports / europeein
Sent from my r00ted dr0id running MIUI
R.I.P. Alexi
Clean bro
Sent from my r00ted dr0id running MIUI
The S6
The S4
my daily.... sorry for the crap cellphone pic
my daily. Cell pic >_
My Toy:
et 3/12
Instagram: VWCorrado91
Sent from my r00ted dr0id running MIUI
Lol, nice. I was trying to take it easy on him. And I thought shocker stickers were cool? I see them alot :x
I hope this kid has cool fender vents too.keep them big brakes cool!
Sent from my r00ted dr0id running MIUI
Ouch guys. For your information, car came with the scoop and at some point it will be restored back to normal.
Trust me I'm not a fan of it either but considering this car had more history behind it then any of yours, I couldn't pass up the chance to own it and bring it back to its original status from when it was featured in euro car and vw power mag.
For those who have been around the dub scene since the 90's, you would know this car is the ABT 3.1l vr6 dubbed "beast from the east" and inspired many corrado owners to live up to it.
Are you the same guys I'm rolling with to dubs on defrost? Can't wait to meet you all
Guru Motorsports / europeein
Last edited by R32R1; 04-05-2012 at 09:13 PM.
I'm game. My Bucket.
GSE - Granite State European
TSP: Team Sad Panda.... When Mods Don't Come Easy
**SOLD** '04 Nogaro "BLüMEäNIE" S4 - APR Tune - APR Carbonio Intake - APR Cat Back Exhaust - JHM Pulley - Cupra Lip - DTH Side Skirts - H&R Sports - Stern 19x9.5 / BB5 LM "Reps" 19x8.5
Guru Motorsports / europeein
I thought that car looked familiar. Have to dig up those old mags and see if I can find it. I think you should keep the scoop. Mad respect for anyone who has preserved a piece of scene history.
As for Marc and cool boy R, don't fead into them. They haven't even put their cars on here. This post is for pictures of local VWs, not bashing other people's rides!
Here's mine. Unfortunately not in this condition any longer. Hoping to do reconstruction soon.


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Biography All Music GuideWikipedia
All Music Guide:
In India, one performer dominates the rap scene: the Lucknow-born Baba Sehgal. Baba Sehgal's rap is far removed from the sting and grit of Public Enemy, Ice-T, or, to take an example of rap transplanted into another language, French wit MC Solaar. Sehgal's safe, inoffensive pop/rap employs a cutesy-pie image with cheeky Hindi lyrics about hobnobbing with Madonna on the telephone. Interjections of a Hinglish nature (a relative of Japlish, the seemingly dyslexic or random plucking of English words in adverts or on bomber jackets or baseball caps in Japan), like "Five star hotel," "James Bond," "Madonna is a very good friend of mine," abound. Superstardom is his self-professed goal. Despite the transparency of his aims, his is a rare instance of non-filmi success in India.
Baba Sehgal is an Indian rapper, film composer, actor and playback singer, known for his works in Indipop, Telugu cinema, Bollywood and Tamil cinema. Brought up in Lucknow, he began his career in the mid-1990s. He is credited as the first Indian rapper to jump on the Indipop bandwagon, and released an album which got frequent airplay on MTV India. Since then he has dominated the Indian rap scene with hits such as 'Thanda Thanda Paani' (a remake of "Ice Ice Baby" by Vanilla Ice), 'Manjula' and 'Dil Dhadke'.^ Dutt, Tushar (13 July 2007). "Baba's back with a bang". The Times of India. Retrieved 6 October 2008. ^ "A sip of Indi-pop". The Indian Express. 18 November 1998. Retrieved 6 October 2008. ^ Abbas, M. Ackbar; John Nguyet Erni; Wimal Dissanayake (2005). Internationalizing Cultural Studies (LINK TO GOOGLE SNIPPET). Blackwell Publishing. ISBN 978-0-631-23623-8. Retrieved 6 October 2008. ^ Hunt, Ken (2003). Vladimir Bogdanov, Chris Woodstra, Stephen Thomas Erlewine, John Bush (eds.), ed. All Music Guide to Hip-hop. Backbeat Books. p. 427. ISBN 978-0-87930-759-2. Retrieved 6 October 2008. ^ Garber, Marjorie; Paul B. Franklin; Rebecca L. Walkowitz (1996). Field Work. Routledge. p. 58. ISBN 978-0-415-91454-3. Retrieved 6 October 2008. ^ "Baba unplugged". The Hindu. 1 March 2008. Retrieved 6 October 2008.
To begin profiling about India's Original Rapstar Baba Sehgal popularly known as the King of Rap, his latest single GOING TO THE GYM has collectively gathered 1 million views followed by another funny investment single MERE PAAS HAI MUTUAL FUND which has been extensively covered by all leading news channels. He has released another single on soundcloud EK PAL WITH AN ANIMAL and his fondness for KISHORE KUMAR is quite evident in this beautiful song. Baba Sehgal is an electrical engineer from G.B.Pant University of agriculture & technology, Pantnagar, Nainital. Sehgal's first album was Dilruba (1990) followed by Alibaba (1991), Thanda Thanda Pani (1992), Main Bhi Madonna (1993), Double Gadbad (1999) and many more albums. He almost always writes, composes and sings the songs in his albums himself. He was the music director of the movie Dance Party (1995) which had a popular song "kapurthala se aaya hoon, tere liye laaya hoon, Orange kurta peela pajama..". He has the distinction of having the first Indian music video ever to be shown on MTV Asia (which was broadcasting out of Hong Kong at the time). He was also the television host of the TV show Superhit Muqabla which was aired on DD2 at primetime. He has also worked as a stage performer.
He was in New York from 2001 to 2005 and when he came home to Mumbai, he released his album 'Welcome to Mumbai', which was his 22nd and latest music album.
He also directed the music for the Bollywood film Bhoot Uncle (2006). He is also anchoring the TV show 'Santa and Banta news unlimited' on Zoom.
In 1998, Sehgal made his acting debut in the Bollywood film Miss 420 alongside Sheeba. He also did playback singing for the film's soundtrack which was released in 1994, four years before the film's theatrical release. In 1999 he acted in the film Double Gadbad playing a double role. He also composed and sang all the songs for the film's soundtrack. In 2009, he played a role in the SAB TV comedy series Jugni Chali Jalandhar. In 2011 he appeared in Rang Badalti Odhani on Star One. Sehgal also making his Telugu film debut in a Heroine centric magnum opus movie Rudhramadevi, Anushka Shetty in the lead. It will be directed by National award winner Gunasekhar. Baba Sehgal has been signed to play a major negative role in another Telugu film titled Overdose.^ Nair, Vinod (14 June 2003). "Arre Baba, he's back!". The Times of India. Retrieved 6 October 2008. ^ Cultures of the Commonwealth. Université de Cergy-Pontoise. 1998. p. 11. Retrieved 6 October 2008. ^ "Baba unplugged". The Hindu. 5 March 2008. Retrieved 6 October 2008. ^ Robertson, Roland; Kathleen E. White (2003). Globalization. Taylor & Francis. p. 234. ISBN 978-0-415-30222-7. Retrieved 6 October 2008. ^ "Dance Party". Bollywood Hungama. Retrieved 6 October 2008. ^ Sharma, Amitabh (26 December 1994). "India's Turning 'Asian Kool' Into Very Hot Sounds Pop music: Record chiefs bet the next global hits will be rap monologues tinged with a Punjabi folk genre known as bhangra". Los Angeles Times. Retrieved 6 October 2008. ^ "Baba Sehgal is back". The Hindu. 21 May 2005. Retrieved 6 October 2008. ^ "Straight Answers". The Times of India. 1 November 2005. Retrieved 6 October 2008. ^ "Baba Sehgal comes back in a new player movement". The Hindu. 26 August 2005. Retrieved 6 October 2008. ^ "Welcome To Mumbai – Baba Saigal". IndiaGlitz. 8 January 2007. Archived from the original on 22 September 2008. Retrieved 6 October 2008. ^ Deladia, Priyanka (15 April 2006). "Straight Answers". The Times of India. Retrieved 6 October 2008. ^ Mulchandani, Amrita (19 May 2008). "'Baba and I are like husband and wife'". The Times of India. Retrieved 6 October 2008. ^ http://zoot.mtsindia.in/artist-Baba%20Sehgal-biography-835
As actorRudramadevi (2014)My Friend Ganesha 3 (2010)Miss 420 (1998)
As playback singerTelugu CinemaRikshavodu (1995)Jalsa (2008)Arya 2 (2009)Adhurs (2010)Arya 2 (2010)Don Seenu (2010)Mr.Perfect (2011)Ragada (2010)Gabbar Singh (2012)Shadow (2013)Om 3D (2013)Action 3D (2013)Adda (2013)Bollywood13B (2009)Miss 420 (1998)Hum Hain Bemisaal (1994)Tamil CinemaBengali Cinema


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Red Hat Extending Financial Division
Red Hat has reinforced its Financial Division and appointed Mark Cook as Vice President for Finance and Controller. Paul Argiry will be joining the company as a Vice President and Treasurer.
Mark Cook joined Red Hat in 2004 and later was promoted to Vice President and Treasurer. According to Red Hat he has over 30 years of experience in enterprise financing and was the Financial Director of various clothing corporations before he joined Red Hat.
Paul Argiry is the new man with Red Hat. Among other things, he will be responsible for the payroll and asset management. Argiry has over 15 years of experience in finance management and worked as a Senior Financial Director with an electronics company before moving.
Both will report directly to Charlie Peters, Red Hat's Chief Financial Officer. Red Hat made another important personnel decision at management level just recently, appointing Michael Chen as Vice President Corporate Marketing as Linux Magazine reported.
Issue 175/2015
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Code.org and College Board Team Reach Out for Talented High School Coders
New partnership will bring more and better CS training to US schools
A Peek Inside TeslaCrypt Ransomware
Criminals offer online help over Tor network
Mumblehard Still Spams
Sophisticated malware is still present on Joomla and WordPress sites around the world.
Launchpad Gets Git
Future versions of Ubuntu's code service will support the popular Git version control system used with Linux and other open source projects.
AMD Posts Code for Linux GPU Driver
New release marks the arrival of AMD's unified driver strategy.
30 Percent of New Server Equipment Goes to Cloud Infrastructure
A new study by IDC charts big changes in the big hardware market.
Windows Might Go Open Source
Azure CTO says Redmond has already considered the unthinkable.
Linux Mint Will Stay with Ubuntu
Lead developer quells rumors that the Debian version is slated for center stage.
Microsoft Frees Code for .NET Build Engine
MSBuild is now just another GitHub project as Redmond continues its path to the light.
Researchers Demonstrate Hack by Heat
Malware could pass data and commands between disconnected computers without leaving a trace on the network.


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You Know It's True: Top 10 Worthless Group Members- By Bossip Staff Categories: Entertainment, Multi, New
There's always that one in the group that we all know is useless but we tolerate anyway. In most cases, the weak links and third wheels quietly fade to black unlike the infamous good-for-nothings on this list who will always be burned into our memories.
Here are the ten most worthless group members ever. Take a look.
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No, this isn't a joke. I mean, it IS a joke, I'm fucking laughing. But there will seriously be a Tetris movie in the near future. A live action one at that. And by "live action" I mean "99% CGI."
"Brands are the new stars of Hollywood," Threshold Entertainment's Larry Kasanoff says. "We have a story behind 'Tetris' which makes it a much more imaginative thing."
First, brands are the new stars of Hollywood? Sure, if you consider all of the cheap, shameless advertising products get in every film nowadays. Reboots are the new stars, you asshat. In other words, movies from 30 years ago are the new stars.
Second, there better be a story behind the movie, because the game you're adapting the movie from HAS NO FUCKING STORY. None. There's no story in place at all. You drop blocks and make solid lines. That's it. If I ever thought Hollywood had run out of ideas, I was completely wrong, because NOW they've fucking run out of ideas.
I'm a writer and I can't possibly fathom any story that would make sense with Tetris involved unless it was The Wizard 2, or Tetris isn't the main character or story. There's no way this is going to be good. I can imagine a movie about something having nothing to do with Tetris or video games in general, and one person walks by an open door and someone is in that room playing the game.
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Composed by Felix Arndt (1889-1918). For piano solo. Original Works. Romantic, American. SMP Level 8 (Early Advanced). Score. Composed 1915. Published by Masters Music Publications Inc. (MT.M3562-SC)
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Composed by Lynn Freeman Olson. Piano. For Piano. Lynn Freeman Olson Classic Solos - Late Elementary Piano. Classical. SMP Level 3 (Early Intermediate); NFMC Level: Elementary Class 2. Solo part. Standard notation. 7 pages. Published by Carl Fischer (CF.P3283)
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This blog is about developing Windows applications using Visual Studio. All postings on this weblog are provided "AS IS" with no warranties, and confer no rights. Use of any samples are subject to the terms specified at http://www.microsoft.com/info/cpyright.htm
Your host Nikola Dudar is a Program Manager in Windows division of Microsoft Corporation. He has been working on Windows Web Services API during Windows 7 and various additions to Visual C++ during VS2005 and VS2008. More details are in LinkedIn profile under Nikola's formal name Mykola Dudar.
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Hello everyone! I am the Program Manager for the Visual C++ Libraries team. The libraries team owns and maintain all VC++ libraries. The most well-known libraries are the C Runtime, Standard C++ library, MFC and ATL. I also closely work with the Platform SDK team, that delivers tools and libraries to build 32bit and 64bit applications and systems for Windows XP and Windows Server 2003. If you have ever requested the VC++ 64bit SDKfor VS2003, you already know that I am going to be the person who talks to you about every issue related to this SDK. I am also a part of the programming model team, which sets what is Microsoft C++ and what can be done using C++. This team consists of representatives from each team in VC++ and I represent Libraries team in our meetings and discussions.
I am joining the community of Microsoft bloggers primarily to share my thoughts and expertise on Visual C++ libraries and C++ itself. In addition to this, I am planning to use this blog to address most often asked questions and problems that users of VC++ libraries are hitting. I will try to re-post most interesting issues reported in VC++ and how these issues can be worked around or resolved.
So enough with introduction and in my next post I am going to talk about have been asked several times. Many our customers and parterns have a question in their mind on what they should use to build 64bit applications that target SP1 of Windows Server 2003 and WindowsXP.
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- Who we are
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Are there any good quality cartoons/movies about the birth of Jesus target to kids ages 2-4? My DD is getting all excited for Christmas but all the shows are about Santa (which we don't do.)
Bri helpmeet to Chaise mama to K(2/07) M(3/09) & A(2/11)
we do not do Santa either. I do not know of any cartoons. We bought the little people nativity scene and re-enacted the Christmas story with my DS. It is still his favorite christmas activity and he actually tells the story very acurately. We started with an abridges version from his children's bible, and now I read the full story. He can quote quite a bit of it now.
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Program shown to work in fight against diabetes
It compares two groups of patients seen in San Diego County clinics. All have the same diabetes diagnoses. The only difference, Philis-Tsimikas said, is that one was seen by Project Dulce's promotoras while the other received more traditional diabetes care.
The study, first published in 2011, showed that the patients who worked with a promotora showed markedly better results than those who received traditional diabetes management. For example, Dulce participants showed, on average, a 7.2 percent drop in total cholesterol compared with a 2.4 percent drop for nonparticipants.
Dr. Edwin Fisher, director of the Peers for Progress program, an international organization that advocates the use of peer educators in diabetes management, said Project Dulce is known for its results.
"I think they've made a real contribution in showing how you can train nonprofessionals to run these programs," Fisher said.
Project Dulce has done training in locations as diverse as Los Angeles, Tijuana and Camden, N.J. Most recently Philis-Tsimikas presented results to doctors in India.
Firoozeh Vali, vice president of research for the New Jersey Hospital Association, said the state sponsored a pilot Project Dulce program at three hospitals in 2006. Today, the state is looking for funds to expand its program to the state's Indian population.
"Our results have actually been a bit better than what they saw in California," Vali said.
Watching interest in the project grow has amazed Philis-Tsimikas.
"I was not convinced, when we started this 15 years ago, that peer educators, who sometimes don't have more than a sixth-grade education, could do something like this … and yet, our results show that they did and they can," Philis-Tsimikas said.
The program also appears to be cost effective. Dr. James Schultz of Neighborhood Healthcare, a community clinic network in San Diego County, said studies have shown that fewer hospital and emergency room visits can save money within one year's time.
"It's a low-cost but very powerful intervention," he said.


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Calhoun County
This 2,225-acre lake is located south of U.S. Hwy. 90 at Grand Ridge and Sneads. Ocheesee has dark tannic water with approximately 90% of its surface area covered by cypress trees. There is access to the lake by taking SR 69 south of Grand Ridge (1.0 mile); turn left on CR 69A (5.9 miles) and travel left on Shady Grove Rd. to the ramp.For local information contact Fisheries Biologists from the Division of Freshwater Fisheries Management, Northwest Regional Office (850-265-3676).
Anglers should exercise caution navigating in the pond due to the many stumps and snags. In the past, this pond has produced some very large bluegill. Try fishing around stumps and snags in 3 to 5 ft or deep holes in the main section of the lake. Best baits include grass shrimp, crickets, wigglers, or various colors of 1/16 to 1/8 oz. beetle spins. Redear sunfish (shellcracker) can also be found in these areas using worms. Try fishing for largemouth bass in the early morning and late afternoon along weedlines and cypress trees. Anglers have reported success fishing for bass using top-water plugs. Best baits include spinner baits, crankbaits, topwater plugs, dark plastic worms or live shiners. Black crappie, good sized warmouth and chain pickerel can also be caught here.
FWC Facts:Programs such as Hooked on Fishing Not on Drugs© have successfully reduced the potential for youth attraction to dangerous lifestyles.
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Calif. prof.'s email reveals shooting plot
SANTA ANA, Calif. Rainer Reinscheid was into his second bottle of wine when he wrote a chilling email titled, "a good plan," detailing how he wanted to get revenge on the people he blamed for his son's suicide.
Reinscheid's son hung himself hours after being disciplined by the assistant principal at his high school in Irvine, setting the university professor on a weekslong downward spiral that authorities say included setting small fires and sending emails in which he vented his anger about school officials.
He wrote about fantasizing about buying a dozen guns, killing 200 University High students, sexually assaulting a school counselor and killing the assistant principal who was the last person to speak to his son, 14-year-old Claas Stubbe.
"I will make him cry and beg, but I will not give him a chance, just like he did to Claas," Reinscheid wrote. "I will make him die, slowly, surely. Next I will set fire to Uni High and try to burn down as much as I can, there should be nothing left that gives them a reason to continue their miserable school."
The emails, which prosecutors say he wrote to himself and to his wife, were obtained by The Associated Press on Wednesday from court filings.
Reinscheid never acted on his most violent musings, but prosecutors charged him with setting a series of arson fires that targeted his late son's school, the assistant principal's home and the park where the boy hanged himself. Five fires erupted between July 1 and July 19 and police apprehended Reinscheid as he tried to start a sixth one on July 24, Irvine police Lt. Julia Engen said.
The emails were discovered on Reinscheid's smartphone by police who were investigating the arsons and were filed by prosecutors in court to support a successful motion to deny bail.
Police believe Reinscheid acted alone and investigators discovered no evidence that he was preparing for a shooting, Engen said.
Reinscheid, who also holds German citizenship, has not been charged with anything related to the content of the emails because they were private communications between Reinscheid and his wife, said Farrah Emami, a district attorney spokeswoman.
Defense attorney Ron Cordova did not return multiple calls for comment. He told the judge in court Tuesday that he didn't want his client to "suffer from a media circus."
In an email to himself in April, while he was on medication to stay awake and drinking his second bottle of wine, Reinscheid wrote he had dreams of burning down the school and killing himself in the place where his son died. He also told his wife he loved her and was sorry if he disappointed her, and asked her to take care of her two children as a single mother.
"I hope you will tell Timmy only one story: Daddy was so sad when Claas passed away, he was just eaten away by his sadness and stopped breathing," he wrote to his wife.
Reinscheid's late son was his child from a first marriage. He also has a step-daughter and son from his second marriage.


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The Detroit News Offers Buy One Get One Auto Show Tickets
DETROIT, Jan. 18 /PRNewswire/ — Beginning Tuesday, January 19 and running through Thursday, January 21 The Detroit News is offering tickets to the North American International Auto Show (NAIAS) at a buy one get one free rate.
(Logo: http://www.newscom.com/cgi-bin/prnh/20081105/CLW096LOGO)
"We're happy to help our readers in this tough economy," said Jon Wolman, Editor and Publisher of The Detroit News. "The Auto Show is a positive way to bring the community together and to support the local auto industry."
Coupons will only run in the print edition of The Detroit News and are valid January 19-21 for general admission only. They can be redeemed at the NAIAS box office.
Detroit Media Partnership, L.P. manages the business functions of the Detroit Free Press and The Detroit News. As the largest newspapers in Michigan, with two of the most visited Web sites in the market, the Free Press and The News combine to reach a weekly audience of 1.9 million people; 50% of adults in this market see our products.* Detroit Media Partnership is also the advertising sales agent for the following suburban Detroit publications: Observer & Eccentric and Mirror Newspapers, Novi News, Milford Times, Northville Record, South Lyon Herald and Livingston County Daily Press & Argus. Gannett Co., Inc., owner of the Free Press, is the general partner in DMP; MediaNews Group, owner of The News, is the limited partner.
*SOURCE: 2009 Scarborough Report — August 2009 — July 2008 — Detroit DMA
SOURCE Detroit Media Partnership


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At Primary Eyecare Optometrics, we're committed to providing the finest full scope vision and eye health care available
It's the same commitment Dr. Leonard Osias started with over 50 years ago. From instrumentation and techniques to this web site, we believe in utilizing state-of-the-art technology in our practice. Employing the highest quality materials and professional staff, we serve the eye care needs of people of all ages. Education is a major component of our practice. Learn more.
Our new location
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We've moved!
We are pleased to announce that we completed our move to our larger facility on Castro Valley Boulevard. The move improves access for a larger number of patients. The larger facility houses state-of-the-art equipment. Better access and better equipment combined with seasoned staff and great service means even better care for all our patients.


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Cheap YQT to Port Hardy flights
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Route information for flights from Thunder Bay Intl. to Port Hardy BC.
1,694.61 miles
YQT - Thunder Bay Intl. Airlines
Browse the most popular airlines servicing the Thunder Bay (YQT) airport. Visit their flights pages to learn more about the most popular routes these carriers fly.


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These suits stand up to chlorine really well. Straps are a little snug.
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I ordered a larger than normal size to accommodate my prego belly like I had done in the past and this suit size it about 2 sizes too small I will reorder as i have had my other one for over 4 years it just now starting to lose the lining and is worn out.
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I bought this suit in hopes that it would last. So far, so good. At first, it seemed tight and uncomfortable. Once I got it on, I realized that the suit fits snugly, yet in a comfortable way. It molds to my frame well, and so far it is the best suit I have ever owned. It is made well, the suit has thick polyester fabric that dries incredibly fast, and it is true to the size. Polyester is the best way to go for heavy use. I am 5"8, 135 pounds, medium torso. I bought a size 36. Finding suits that fit me well has usually been a problem. This suit however, fits me well. I work nearly every day at a highly chlorinated pool. I was worried that buying a nice suit would be a waste of time and money, I believe that this has been a great choice as well as a good find.
I usually wear a 42 suit and since it is polyester i ordered a 44 ... it would not even go over my thighs. not true to size at all
Suit ran small, so we had to order three times in order to get the right fit. It didn't matter, however, because the prompt service and delivery had each suit at our home within three days.
Used to wear these suits years ago and loved them. Just got back to teaching swimming lessons and needed a high quality/durable suit that would hold up well to the long hours in high chlorine. Suit is great, runs small. I ordered a size up but is still a bit snug. Once you're in the water a bit it's not that noticeable but was surprised it fit the way it did even with ordering up a size, didn't remember them being this way years ago when the poly suits first came out. Just be mindful of the sizing when ordering because it's a great suit at an excellent price. Well worth it.
Always order size 26 for my daughter in all brands and always fits EXCEPT this lifeguarding suit- she could not even get it onHad to return it-
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The unique spelling of Dolfin parallels their unique style. Dolfin was founded fifty years ago with a focus on lifeguard suits and has expanded over the years into the competition market. Recently, Dolfin has developed a niche in the practice suit market with their fun, colorful, and long-lasting Uglies line. The Uglies line appeals to girls of all ages and is perfect for competitive and recreational swimmers. Dolfin also has crazy training suits called Dragsters, wild-printed Winner jammers and briefs for guys. Along with their other swim divisions, Ocean Racing and Aquashape, the Dolfin team has everyone covered!