text:
CHICAGO, July 14, 2010 -- NYSE Arca, a unit of NYSE Euronext (NYX), has filed with the Securities and Exchange Commission to introduce a new price collar designed to safeguard the execution of market orders. The new price collar will be introduced on July 15, 2010 and is the latest in a series of steps implemented to improve market practices and structure since the "flash crash" of May 6, 2010.
"The market-order collar is an additional protection that complements those already in place and addresses a specific issue highlighted by the flash crash -- market orders that were executed at anomalous prices in electronic markets. The new collar is designed to help limit potential harm from extreme market volatility by preventing trades from occurring a specified percentage away from the last trade price," said Joseph Mecane, Executive Vice President and Co-Head of U.S. Listings and Cash Execution. "We will continue working closely with the SEC, other markets and market participants toward the goals of further strengthening the markets' safety net and rebuilding investor confidence in our national market system."
The new collar will prevent market orders to buy stock from executing or routing to another trading venue at a price above the collar. Conversely, market orders to sell will not execute or route at a price below the trading collar. The collar for issues priced $25 or less will be 10 percent above or below the last trade price; for issues priced above $25 up to and including $50, the collar will be 5 percent; and for issues above $50, the collar will be 3 percent. These limits also will help prevent erroneous trades from inadvertently triggering the individual-stock circuit breakers introduced last month, and are consistent with those in the newly implemented rules concerning the cancellation of erroneous trades.
Additional details of the new measure, including trading examples, are in the NYSE Arca rule filed with the SEC, linked here: http://apps.nyse.com/commdata/pub19b4.nsf/docs/F9706A0475E6BEAF85257760005AC153/$FILE/NYSEArca-2010-67.pdf
In just over two months since May 6, the following corrective measures have been implemented by the markets in concert with the SEC:
• A pilot program of circuit breakers for individual issues was first rolled out on June 11 for stocks in the Standard & Poor's 500.
• An expansion of the above pilot program to cover 344 exchange traded products plus all stocks in the Russell 1000 index is planned for later this month, pending SEC approval.
• All markets have proposed amendments to existing rules concerning clearly erroneous trades, to make the cancellation of such trades -- when they occur in connection with an individual stock circuit breaker -- transparent and predictable for market participants.
• NYSE Arca has revised its market order routing to further enhance its interaction with the New York Stock Exchange when a Liquidity Replenishment Point has been reached and other individual-stock safeguards imposed by primary markets.
About NYSE Euronext
NYSE Euronext (NYX) is a leading global operator of financial markets and provider of innovative trading technologies. The company's exchanges in Europe and the United States trade equities, futures, options, fixed-income and exchange-traded products. With approximately 8,000 listed issues (excluding European Structured Products), NYSE Euronext's equities markets – the New York Stock Exchange, NYSE Euronext, NYSE Amex, NYSE Alternext and NYSE Arca – represent one-third of the world's equities trading, the most liquidity of any global exchange group. NYSE Euronext also operates NYSE Liffe, one of the leading European derivatives businesses and the world's second-largest derivatives business by value of trading. The company offers comprehensive commercial technology, connectivity and market data products and services through NYSE Technologies. NYSE Euronext is in the S&P 500 index, and is the only exchange operator in the S&P 100 index and Fortune 500. For more information, please visit: http://www.nyx.com.
Cautionary Note Regarding Forward-Looking Statements
This press release may contain forward-looking statements, including forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Such forward-looking statements include, but are not limited to, statements concerning NYSE Euronext's plans, objectives, expectations and intentions and other statements that are not historical or current facts. Forward-looking statements are based on NYSE Euronext's current expectations and involve risks and uncertainties that could cause actual results to differ materially from those expressed or implied in such forward-looking statements. Factors that could cause NYSE Euronext's results to differ materially from current expectations include, but are not limited to: NYSE Euronext's ability to implement its strategic initiatives, economic, political and market conditions and fluctuations, government and industry regulation, interest rate risk and U.S. and global competition, and other factors detailed in NYSE Euronext's reference document for 2009 ("document de référence") filed with the French Autorité des Marchés Financiers (Filed on April 22, 2010 under No. D.10-0304), 2009 Annual Report on Form 10-K and other periodic reports filed with the U.S. Securities and Exchange Commission or the French Autorité des Marchés Financiers. In addition, these statements are based on a number of assumptions that are subject to change. Accordingly, actual results may be materially higher or lower than those projected. The inclusion of such projections herein should not be regarded as a representation by NYSE Euronext that the projections will prove to be correct. This press release speaks only as of this date. NYSE Euronext disclaims any duty to update the information herein.


text:
Celebrity Buzz
Your daily dose of hot news & gossip.
Posts filtered on Tag
Joanna Krupa considering freezing her eggs
Model-turned-reality TV star Joanna Krupa is considering freezing her eggs to make sure she can become a mother one day. The 34-year-old "The Real Housewives of Miami" star is committed to starting a family with her businessman husband Romain Zago, but she fears it will be years before she is ready to settle down. She […] [Read More]
Joanna Krupa used laxatives to lose weight
Model-turned-reality TV star Joanna Krupa struggled with weight loss at the beginning of her fashion career and turned to laxatives to help her shed the pounds. The 34-year-old "Real Housewives of Miami" star feared not maintaining a skinny frame would cost her her job as a top model and came up with more extreme ways [Read More]
Reality star Joanna Krupa and husband attend sex therapy
Model-turned-reality TV star Joanna Krupa and her husband Romain Zago are attending sex therapy sessions in a bid to spice up their two-month marriage. The couple has been together for over six years and wed in June, after briefly splitting last year over Zago's lack of interest in sex. And now Krupa, who stars in […] [Read More]
Joanna Krupa weds in California
Model-turned-reality TV star Joanna Krupa has wed her fiancé Romain Zago in Carlsbad, Calif. The couple exchanged vows on Thursday afternoon at the Park Hyatt Aviara Resort in front of 150 guests. "The Real Housewives of Miami" star and the entrepreneur have been together for six years, but briefly split in November after Krupa went […] [Read More]
Joanna Krupa is now single
Model Joanna Krupa has called off her lengthy engagement to Florida nightclub impresario Romain Zago. The reality TV star ended their five-and-a-half-year engagement citing Zago's commitment issues as the reason behind the split. She told In Touch magazine, "We got in a huge fight, and I just pretty much got fed up with it. He […] [Read More]
Supermodel-turned-reality TV star Joanna Krupa is threatening to sue the editors of a gossip website over allegations suggesting she's a high-class call girl. The former "Dancing With the Stars" contestant, who is a new member of "America's Real Housewives" team, was appalled to read an article on TheDirty.com ,which linked her to a prostitute ring […] [Read More]

subset: commoncrawl


text:
ACS to host Relay for LifeWritten by Zach Davis | | firstname.lastname@example.com
The American Cancer Society will be holding the Relay for Life on June 11-12 at Maumee High School Stadium.
The 18-hour event will raise money to improve the quality of life for cancer patients and the outlook for those who have yet to be diagnosed. It begins June 11 at 2 p.m. and lasts until June 12 at 8 a.m.
The event will be open to the public for fundraising, food and live entertainment as the relay takes place. So far, 32 teams have registered to compete in the Relay for Life.
"We get a bunch of people to form teams and try to raise money throughout the year, but this is the main event," Entertainment and Activities Committee Member Don Zellers said. "Each team does fundraisers during the course of the year. The teams of 10 to 15 people each try to have a person on the track for the entire 18 hours and during that time we will have entertainment and a bunch of activities going on for the people."
Entertainment opportunities include dancing and a cornhole tournament, as well as other children's entertainment and games including a bouncy house. Live entertainment includes Curtice Markley and East River Drive.
Typically, the American Cancer Society honors about three cancer survivors per year as "Heroes for Hope." Sue Robinson is one of those honorees this year along with her husband Bob, who is being honored as the second-annual "Honorary Caregiver." The Robinsons have been involved with the Relay for Life for about 10 years. Other honorees include Santina Delucia and Diane Schult.
The Robinsons had an extremely difficult year in 1994 when their daughter, Teri Sue, was diagnosed with squamous cell carcinoma of the throat and tongue just one month before Sue was diagnosed with Stage II breast cancer.
"It was just a long haul for about eight months," Bob Robinson said. "They both went through chemotherapy and radiation. For the next eight months after they were diagnosed it was running one or the other to the hospital every day for those treatments."
After a 25-hour operation, Teri Sue's condition improved but the cancer came back later in the year and spread to her bones. She passed away 10 days before Christmas in 1994 at just 27 years old.
"She came through the operation really well," Bob Robinson said. "Everything was going really well until a few months later when we found out the cancer had spread. Eventually Teri, ended up in the hospital and the point came where she was diagnosed terminal. We brought her home and she spent the last couple months with us."
Meanwhile, Sue continued to go to work through treatments and, as Bob described, "was more concerned about her daughter than herself." She was deemed cancer-free in early 1995 and has remained so ever since.
As his wife and daughter battled cancer, Bob faced depression and began to see a psychologist to help deal with his situation.
"It was killing me to not know what was going on with both women I love," Bob Robinson said. "I knew I needed some help so I got it and that was the best thing I ever did."
Sue joined her husband last year in retirement after remaining at her job for 42 years. They now both travel annually to the Florida Keys to celebrate Sue beating cancer, which they have done for the past 16 years.
"We survived all the stuff that happened to us," Bob Robinson said. "We still feel pretty blessed that we have a beautiful home and a lot of friends and family. I don't know how people do it that don't have faith, friends and family. It's hard enough of a struggle with that stuff. We had a lot of support and that's a big deal."


text:
This Page Last Updated on 03/10/2006
Main Menu
|Purpose and Leadership
Purpose and Leadership
The following are instructions on how to organize and run the monthly PLC meeting. The purpose of the monthly PLC Meeting is to share information between the Patrols and the PLC and to review and plan the Troop's Program.
WHO IS IN CHARGE: The SPL runs the meeting.
WHO ATTENDS: The SPL, ASPLs, Patrol Leaders (Including the Patrol Leader of the Senior Patrol), and Troop Guides attend the PLC Meeting and have a vote. The Troop Scribe attends and takes the minutes of the meeting. The Troop Scribe types up the minutes of the PLC Meeting and distributes them to members of the PLC. The Scoutmaster also attends the PLC Meeting as an Advisor. Patrol Advisors, Troop Committee Chair, and ASMs may attend. Assistant Patrol Leaders may attend if they want, but may not vote unless their Patrol Leader is not attending the meeting. If a Patrol's Patrol Leader and Assistant cannot attend the PLC Meeting, then the Patrol Leader is responsible to find another Patrol member to attend the PLC Meeting to represent the Patrol as a voting member.
Before the PLC Meeting
The following steps should be done prior to holding the monthly PLC Meeting:
Back to Top
Running the PLC Meeting
The following steps should be followed during the PLC Meeting:
Back to Top
The following steps will be taken after the PLC meeting:
Back to Top
To Download a copy of this How To, right click the link below and use "Save Target As." Download the document and open it on your computer. Directly downloading the document from this link does not work well.


text:
The present invention includes interferon-tau (IFNT) pharmaceutical compositions useful for oral administration to treat cancers, autoimmune disorders (particularly multiple sclerosis), cell proliferative disorders and viral disease.
1. In a method of treating a disease condition in a mammal responsive to treatment by interferon-tau (IFNτ), an improvement comprising orally administering a therapeutically-effective amount of IFNτ.
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. The method of
11. The method of
12. The method of
13. The method of
14. The method of
15. A method of treating an autoimmune disorder in a subject, comprising
orally administering a therapeutically-effective amount of interferon-tau (IFNτ) to said subject.
16. The method of
17. A method of
18. The method of
19. The method of
[0001] This application is a continuation of application Ser. No. 08/616,904 filed Mar. 15, 1996, now allowed, which is a continuation-in-part of application Ser. No. 08/406,190 filed Mar. 16, 1995, now U.S. Pat. No. 5,906,816; and a continuation-in-part of application Ser. No. 08/438,753 filed May 10, 1995, now U.S. Pat. No. 5,705,363; which is a continuation-in-part of application Ser. No. 08/139,891 filed Oct. 19, 1993, now abandoned; which is a continuation-in-part of application Ser. No. 07/847,741 filed Mar. 9, 1992; which is a continuation-in-part of application Ser. No. 07/318,050 filed Mar. 2, 1989, now abandoned and said application Ser. No. 08/139,891 filed Oct. 19, 1993 is a continuation-in-part of application Ser. No. 07/969,890 filed Oct. 30, 1992, now abandoned; all of which are incorporated herein by reference.
[0002] This work was supported in part by grant number AI 25904 awarded by the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.
[0003] The present invention relates to orally-administered pharmaceutical compositions containing interferon-tau and methods of uses thereof.
[0004] Ausubel, F. M., et al., in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media, Pa. (1988).
[0005] Bartol, F. F., et al., Biol. Reprod. 32:681-693 (1985).
[0006] Bayne, M. L., et al., Gene 66:235 (1988).
[0007] Bazer, F. W., et al., J. Animal Sci. 57(Supp. 2):425 (1983).
[0008] Bazer, F. W., et al., J. Reproduction and Fertility 76:841 (1986).
[0009] Bazer, F. W., et al., PCT Application publication No. WO 10 94/10313, published May 11, 1994.
[0010] Beames, et al., Biotechniques 11:378 (1991).
[0011] Benoit, P., et al., J. Immunol. 150(3):707 (1993).
[0012] Blatt, L. M., et al., U.S. Pat. No. 5,372,808, issued Dec. 13, 1994.
[0013] Bonnem, E. M., et al., J. Bio. Response Modifiers 3:580 (1984).
[0014] Clayman, C. B. , Ed., AMERICAN MEDICAL ASSOCIATION ENCYCLOPEDIA OF MEDICINE(Random House, New York, N.Y.), 1991.
[0015] Cross, J. C., and Roberts, R. M., Proc. Natl. Acad. Sci. USA 88:3817-3821 (1991).
[0016] Davis, G. L., et al., N. England J. Med. 321:1501 (1989).
[0017] Davis, G. L., et al., Theriogenology 38:867 (1992).
[0018] Day, M. J., et al., Clin. Immunol. Immunopathol. 35(1):85-91 (1985).
[0019] Degre, M., Int. J. Cancer 14:699 (1974).
[0020] DeMaeyer, E. , et al., in INTERFERONS AND OTHER REGULATORY CYTOKINES, John Wiley and Sons, New York (1988).
[0021] Dusheiko, G. M., et al., J. Hematology 3(Suppl. 2):S199 (1986).
[0022] Ecker, D. J., et al., J. Biol. Chem. 264:7715-7719 (1989).
[0023] Ernst, J. F., DNA 5:483 (1986).
[0024] Familetti, P. C., et al., Meth. Enzymol. 78:387 (1981).
[0025] Feher, Z., et al., Curr. Genet. 16:461 (1989).
[0026] Fent, K. and G. Zbinden, Trnds. Pharm. Sci. 56:1-26 (1987).
[0027] Figuero, F., et al., Immunogenetics 15(4):399-404 (1982).
[0028] Finter, N. B., et al., Drugs 42(5):749 (1991).
[0029] Fritz, R. B., et al., J. Immunol. 130(3):1024-1026 (1983).
[0030] Gelvin, S. B. and R. A. Schilperoot, Plant Molecular Biology (1988).
[0031] Gnatek, G. G., et al., Biol. Reprod. 41:655-664 (1989).
[0032] Godkin, J. D., et al., J. Reprod. Fertil. 65:141-150 (1982).
[0033] Hansen, P. J., et al., U.S. Pat. No. 4,997,646, issued Mar. 5, 1991.
[0034] Harlow, E., et al., in ANTIBODIES : A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).
[0035] Hitzeman, R. A., et al., U.S. Pat. No. 4,775,622, issued Oct. 4, 1988.
[0036] Helmer, S. D., et al., J. Reprod. Fert. 79:83-91 (1987).
[0037] IFNβ Multiple Sclerosis Study Group, Neurology 43(4):655 (1993).
[0038] Imakawa, K., et al., Nature 330:377-379 (1987).
[0039] Imakawa, K., et al., Mol. Endocrinol. 3-:127 (1989).
[0040] Johnson, H. M., et al., Sci. Am. 270(5):40-47 (1994).
[0041] Kashima, H., et al., Laryngoscope 98:334 (1988).
[0042] Kemppainen, R. J., and Clark, T. P., Vet. Clin. N. Am. Small Anim. Pract. 24(3):467-476 (1994).
[0043] Klein, J., et al., Immunogenetics 17:553 (1983).
[0044] Kotzin, B. L., et al., J. Exp. Med. 265:1237 (1987).
[0045] Kristensen, A. T., et al., J. Vet. Intern. Med. 8(1):36-39 (1994).
[0046] Lider, et al., J. Immunol., 142:148-752 (1989).
[0047] Ludwig, D. L., et al., Gene 132:33 (1993).
[0048] Martal, J., et al., J. Reprod. Fertil. 56:63-73 (1979).
[0049] Martin, E. W., in DISPENSING OF MEDICATION: A PRACTICAL MANUAL ON THE FORMULATION AND DISPENSING OF PHARMACEUTICAL PRODUCTS Mack Publishing Co., Easton, Pa. (1976).
[0050] Mullis, K. B., U.S. Pat. No. 4,683,202, issued Jul. 28, 1987.
[0051] Mullis, K. B., et al., U.S. Pat. No. 4,683,195, issued Jul. 28, 1987.
[0052] Oeda, K., et al., U.S. Pat. No. 4,766,068, issued Aug. 23, 1988.
[0053] Oldham, R. K., Hospital Practice 20:71 (1985).
[0054] Pearson, W. R. and Lipman, D. J., PNAS 85:2444-2448 (1988).
[0055] Pearson, W. R., Methods in Enzymology 1:63-98 (1990).
[0056] Pontzer, C. H., et al., Cancer Res. 51:5304 (1991).
[0057] Quesada, J. R., et al., N. England J. Med. 310:15 (1984).
[0058] Reilly, P. R., et al., in BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).
[0059] Roberts, R. M., et al., Endocrin. Rev. 13:432-452 (1992).
[0060] Rutter, W. J., et al., U.S. Pat. No. 4,769,238, issued Sep. 6, 1988.
[0061] Sabin, E., et al., Bio/Technology 1:705-709 (1989).
[0062] Sambrook, J., et al., in MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
[0063] Shaw, K. J., et al., DNA 7:117 (1988).
[0064] Shen, L. P., et al., Sci. Sin. 29:856 (1986).
[0065] Smith, P. K., et al., Anal. Biochem. 150:76 (1985).
[0066] Stewart, H. J., et al., J. Endocrinol. 115:R13 (1987).
[0067] Weiner, H., et al., Ann. Rev. Immunol. 12:809-837 (1994).
[0068] Weinstock-Guttman, B., et al., Ann. Neurol. 37:7-15 (1995).
[0069] Werner, L. L., et al., Vet. Immunol. Immunopathol. 8(1-2):183-192 (1985).
[0070] Whaley, A. E., et al., J. Biol. Chem. 269(14):10864-10868 (1994).
[0071] Wilson, et al., Biology of Reproduction 20(Supp. 1):101A, Abstract (1979).
[0072] Wraith, D. C., et al., Cell 5:247 (1989).
[0073] Wu, D. A., et al., DNA 10:201 (1991).
[0074] Zamvil, S. S., and Steinman, L., Ann. Rev. Immunol. 8:579-621 (1990).
[0075] Conceptus membranes, or trophectoderm, of various mammals produce biochemical signals that allow for the establishment and maintenance of pregnancy (Bazer, et al., 1983). One such protein, ovine trophoblast protein-one (oTP-1), was identified as a low molecular weight protein secreted by sheep conceptuses between days 10 and 21 of pregnancy (Wilson, et al., 1979; Bazer, et al., 1986). The protein oTP-1 was shown to inhibit uterine secretion of prostaglandin F2-alpha, which causes the corpus luteum on the ovary to undergo physiological and endocrinological demise in nonpregnant sheep (Bazer, et al., 1986). Accordingly, oTP-1 has antiluteolytic biological activity. The primary role of oTP-1 was assumed to be associated with the establishment of pregnancy.
[0076] oTP-1 was subsequently found to (i) exhibit limited homology (50-70%) with interferon alphas (IFNα) of various species (Imakawa, et al., 1987), and (ii) bind to a Type I interferon receptor (Stewart, et al., 1987). Despite some similarities with IFNα, oTP-1 has several features that distinguish it from IFNα including the following: oTP-1's role in reproductive biochemistry (other interferons are not known to have any role in the biochemical regulation of reproductive cycles), oTP-1's cellular source—trophoblast cells (IFNα is derived from lymphocyte cells), oTP-1's size—172 amino acids (IFNα is typically about 166 amino acids), and oTP-1 is weakly inducible by viruses (IFNα is highly inducible by viruses). The International Interferon Society recognizes oTP-1 as belonging to an entirely new class of interferons which have been named interferon-tai (IFNτ). The Greek letter τ stands for trophoblast.
[0077] The interferons have been classified into two distinct groups: type I interferons, including IFNα, IFNβ, and IFNω (also known as IFNαII); and type II interferons, represented by IFNγ (reviewed by DeMaeyer, et al., 1988). In humans, it is estimated that there are at least 17 IFNα non-allelic genes, at least about 2 or 3 IFNβ non-allelic genes, and a single IFNγ gene.
[0078] IFNα's have been shown to inhibit various types of cellular proliferation. IFNα's are especially useful against hematologic malignancies such as hairy-cell leukemia (Quesada, et al., 1984). Further, these proteins have also shown activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinary bladder tumors and ovarian cancers (Bonnem, et al., 1984; Oldham, 1985). The role of interferons and interferon receptors in the pathogenesis of certain autoimmune and inflammatory diseases has also been investigated (Benoit, et al., 1993).
[0079] IFNα's are also useful against various types of viral infections (Finter, et al., 1991). Alpha interferons have shown activity against human papillomavirus infection, Hepatitis B, and Hepatitis C infections (Finter, et al., 1991; Kashima, et al., 1988; Dusheiko, et al., 1986; Davis, et al., 1989).
[0080] In addition, type I interferons are useful in treating autoimmune diseases such as multiple sclerosis (MS). In fact, IFNβ has been tested and approved by the U.S. Food and Drug Administration (FDA) as an MS therapy.
[0081] Significantly, however, the usefulness of IFNα's has been limited by their toxicity: use of interferons in the treatment of cancer, autoimmune disorders and viral disease has resulted in serious side effects, such as fever, chills, anorexia, weight loss, and fatigue (Pontzer, et al., 1991; Oldham, 1985). These side effects often require (i) the interferon dosage to be reduced to levels that limit the effectiveness of treatment, or (ii) the removal of the patient from treatment. Such toxicity has reduced the usefulness of these potent antiviral and antiproliferative proteins in the treatment of debilitating human and animal diseases.
[0082] The present invention provides methods of treatment for cancer, autoimmune diseases (such as MS) and for inhibiting cellular proliferation and viral infection. These methods do not have the toxic side effects associated with currently-used therapies, and employ a convenient route of administration.
[0083] In one aspect, the present invention includes an improvement in a method of treating a disease condition in a mammal (e.g., mouse, dog or human) responsive to treatment by interferon-tau (IFNτ). The improvement comprises orally administering a therapeutically-effective amount of IFNτ. The orally-administered IFNτ is preferably ingested by the mammal. In a general embodiment, the IFNτ is orally-administered at a dosage of between about 1×105 and about 1×108 units per day, preferably at a dosage of between about 1×106 and about 1×107 units per day. The IFNτ may be, for example, ovine IFNτ (OvIFNτ), e.g., a polypeptide having the sequence represented as SEQ ID NO: 2, or a human IFNτ (HuIFNτ), e.g., a polypeptide having the sequence represented as SEQ ID NO: 4 or SEQ ID NO: 6.
[0084] In one embodiment, the disease condition is an immune system disorder, such as an autoimmune disorder (e.g., multiple sclerosis (MS), type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, allergies or psoriasis). MS is particularly amenable to treatment using the methods of the present invention.
[0085] In another embodiment, the disease condition is a cell proliferation disorder, such as a cancer (e.g., hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous T cell lymphoma, and glioma).
[0086] In yet another embodiment, the disease condition is a viral disease (e.g., hepatitis A, hepatitis B, hepatitis C, non-A, non-B, non-C hepatitis, Epstein-Barr viral infection, HIV infection, herpes virus (EB, CML, herpes simplex), papilloma, poxvirus, picorna virus, adeno virus, rhino virus, HTLV I, HTLV II, and human rotavirus).
[0087] In another aspect, the invention includes a method of treating an autoimmune disorder in a subject (e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNτ) to the subject. The orally-administered IFNτ is preferably ingested by the subject. Examples of autoimmune conditions amenable to treatment, dosages, and sources of IFNτ are as presented above.
[0088] The invention also includes a method of decreasing the severity or frequency of a relapse of multiple sclerosis (MS) in a human suffering from MS, by orally administering a therapeutically-effective amount of interferon-tau (IFNτ) to the human. Examples of dosages and sources of IFNτ are as presented above.
[0089] In another aspect, the invention includes a method of treating a cell proliferation disorder in a subject (e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNτ) to the subject. The orally-administered IFNτ is preferably ingested by the subject. Examples of cell proliferation disorders amenable to treatment, dosages, and sources of IFNτ are as presented above.
[0090] In still another aspect, the invention includes a method of treating a viral disease in a subject (e.g., a human subject), by orally administering a therapeutically-effective amount of interferon-tau (IFNτ) to the subject. The orally-administered IFNτ is preferably ingested by the subject. Examples of viral diseases amenable to treatment, dosages, and sources of IFNτ are as presented above.
[0091] A further aspect of the invention includes a method of enhancing fertility in a female mammal (e.g., a human female), by orally administering a therapeutically-effective amount of interferon-tau (IFNτ) to the mammal. Examples of dosages and sources of IFNτ are as presented above.
[0092] These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.
[0093]FIG. 1 shows the amount of OvIFNτ in NZW mouse sera after administration by either oral feeding (filled bars) or i.p. injection (open bars) as measured using an anti-viral assay.
[0094]FIGS. 2A, 2B and 2C show the prevention of chronic-relapsing experimental allergic encephalomyelitis (EAE) in SJL mice by orally-administered (FIG. 2C) and i.p.-injected (FIG. 2B) IFNτ as compared with mice receiving no treatment (FIG. 2A).
[0095]FIGS. 3A, 3B and 3C show sections of mouse spinal cord stained with cresyl violet for detection of lymphocyte infiltration from EAE-induced animals receiving either no IFNτ treatment (FIG. 3A), OvIFNτ treatment by i.p. injection (FIG. 3B) or OvIFNτ treatment by oral feeding (FIG. 3C).
[0096]FIG. 4 shows induction of IL-10 by either single-dose or prolonged IFNτ treatment administered by i.p. injection or oral feeding.
[0097]FIG. 5 shows relapses of EAE in SJL mice following removal of IFNτ treatment.
[0098]FIG. 6 shows ELISA detection of anti-OvIFNτ antibodies in the sera of OvIFNτ-treated mice following i.p. injection or oral feeding of OvIFNτ.
[0099] SEQ ID NO: 1 is the nucleotide sequence of a synthetic gene encoding ovine interferon-τ (OvIFNτ). Also shown is the encoded amino acid sequence.
[0100] SEQ ID NO: 2 is an amino acid sequence of a mature OvIFNτ protein.
[0101] SEQ ID NO: 3 is a synthetic nucleotide sequence encoding a mature human interferon-τ (HuIFNτ) protein.
[0102] SEQ ID NO: 4 is an amino acid sequence for a mature HuIFNτ1 protein.
[0103] SEQ ID NO: 5 is the nucleotide sequence, excluding leader sequence, of genomic DNA clone HuIFNτ3, a natural HuIFNτ gene.
[0104] SEQ ID NO: 6 is the predicted amino acid sequence of a mature human IFNτ protein encoded by HuIFNτ3, encoded by the sequence represented as SEQ ID NO: 5.
[0105] I. Definitions
[0106] Interferon-τ refers to any one of a family of interferon proteins having at least one characteristic from each of the following two groups of characteristics: (i) (a) anti-luteolytic properties, (b) anti-viral properties, (c) anti-cellular proliferation properties; and (ii) about 45 to 68% amino acid homology with α-Interferons and greater than 70% amino acid homology to known IFNτ sequences (e.g., Ott, et al., 1991; Helmer, et al., 1987; Imakawa, et al., 1989; Whaley, et al., 1994; Bazer, et al., 1994). Amino acid homology can be determined using, for example, the LALIGN program with default parameters. This program is found in the FASTA version 1.7 suite of sequence comparison programs (Pearson and Lipman, 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, Va.). IFNτ can be obtained from a number of sources including cows, sheep, ox, and humans.
[0107] An interferon-τ polypeptide is a polypeptide having between about 15 and 172 amino acids derived from an interferon-τ amino acid coding sequence, where said 15 to 172 amino acids are contiguous in native interferon-τ. Such 15-172 amino acid regions can also be assembled into polypeptides where two or more such interferon-τ regions are joined that are normally discontinuous in the native protein.
[0108] Treating a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
[0109] II. Interferon-tau (IFNτ)
[0110] A. Introduction
[0111] The first IFNτ to be identified was ovine IFNτ (OvIFNτ). Several isoforms of the 18-19 kDa protein were identified in conceptus (the embryo and surrounding membranes) homogenates (Martal, et al., 1979). Subsequently, a low molecular weight protein released into conceptus culture medium was purified and shown to be both heat labile and susceptible to proteases leg (Godkin, et al., 1982). OvIFNτ was originally called ovine trophoblast protein-one (oTP-1) because it was the primary secretory protein initially produced by trophectoderm of the sheep conceptus during the critical period of maternal recognition in sheep. One isolate of mature OvIFNτ is 172 amino acids in length (SEQ ID NO: 2).
[0112] IFNτs with similar characteristics and activities have been isolated from other ruminant species including cows and goats (Bartol, et al., 1985; Gnatek, et al., 1989; Helmer, et al., 1987; and Imakawa, et al., 1989). Bovine IFNτ (BoIFNτ) and OvIFNτ have (i) have similar functions in maternal recognition of pregnancy, and (ii) share a high degree of amino acid and nucleotide sequence homology between mature proteins. The nucleic acid sequence homology between OvIFNτ and BoIFNτ is 76.3% for the 5′ non-coding region, 89.7% for the coding region, and 91.9% for the 3′ non-coding region. The amino acid sequence homology is 80.4%.
[0113] Antisera to all the IFNτs cross-react. This is not unexpected since the species specific forms of IFNτ are more closely homologous to each other than to the IFNsα from the identical species (Roberts, et al., 1992). Relative to other interferons, OvIFNτ shares about 45 to 68% amino acid homology with Interferon-α and the greatest sequence similarity with the interferon-ωs (IFNωs) of about 68%.
[0114] While IFNτ displays many of the activities classically associated with type I IFNs (see Table 1, above), considerable differences exist between it and the other type I IFNs. The most prominent difference is its role in pregnancy, detailed above. Also different is viral induction. All type I IFNs, except IFNτ, are induced readily by virus and dsRNA (Roberts, et al., 1992). Induced IFNα and IFNβ expression is transient, lasting approximately a few hours. In contrast, IFNτ synthesis, once induced, is maintained over a period of days (Godkin, et al., 1982). On a per-cell basis, 300-fold more IFNτ is produced than other type I IFNs (Cross and Roberts, 1991).
[0115] Other differences may exist in the regulatory regions of the IFNτ gene. For example, transfection of the human trophoblast cell line JAR with the gene for bovine IFNτ resulted in antiviral activity while transfection with the bovine IFNβ gene did not. This implies unique transacting factors involved in IFNτ gene expression. Consistent with this is the observation that while the proximal promoter region (from 126 to the transcriptional start site) of IFNτ is highly homologous to that of IFNα and IFNβ; the region from −126 to −450 is not homologous and enhances only IFNτ expression (Cross and Roberts, 1991). Thus, different regulatory factors appear to be involved in IFNτ expression as compared with the other type I IFNs.
[0116] IFNτ expression may also differ between species. For example, although IFNτ expression is restricted to a particular stage (primarily days 13-21) of conceptus development in ruminants (Godkin, et al., 1982), preliminary studies suggest that the human form of IFNτ is constitutively expressed throughout pregnancy (Whaley, et al., 1994).
[0117] B. Production of IFNτ
[0118] IFNτ polypeptides suitable for use in the methods of the present invention may produced in any of a number of ways. For example, they may be purified from animal tissues in which they are expressed, synthesized using a peptide synthesizer or produced recombinantly.
[0119] Recombinant IFNτ protein may be produced from any selected IFNτ polynucleotide fragment using a suitable expression system, such as bacterial or yeast cells. The isolation of IFNτ nucleotide and polypeptide sequences is described in Bazer, et al. (1994). For example, Bazer, et al., describe the identification and isolation of a human IFNτ gene. A synthetic nucleotide sequence encoding a mature human interferon-τ (HuIFNτ) protein is presented herein as SEQ ID NO: 3. SEQ ID NO: 4 is the corresponding amino acid sequence for a mature HuIFNτ1 protein. SEQ ID NO: 5 is the nucleotide sequence, excluding leader sequence, of genomic DNA clone HuIFNτ3, a natural HuIFNτ gene, and SEQ ID NO: 6 is the predicted amino acid sequence of a mature human IFNτ protein encoded by the sequence represented as SEQ ID NO: 5.
[0120] To make an IFNτ expression vector, an IFNτ coding sequence (e.g, SEQ ID NO: 1 ) is placed in an expression vector, e.g., a bacterial expression vector, and expressed according to standard methods. Examples of suitable vectors include lambda gt11 (Promega, Madison Wis.); pGEX (Smith, et al., 1985); pGEMEX (Promega); and pBS (Stratagene, La Jolla Calif.) vectors. Other bacterial expression vectors containing suitable promoters, such as the T7 RNA polymerase promoter or the tac promoter, may also be used. Cloning of the OvIFNτ synthetic polynucleotide into a modified pIN III omp-A expression vector is described in the Materials and Methods.
[0121] For the experiments described herein, the OvIFNτ coding sequence present in SEQ ID NO: 1 was cloned into a vector, suitable for transformation of yeast cells, containing the methanol-regulated alcohol oxidase (AOX) promoter and a Pho1 signal sequence. The vector was used to transform P. pastoris host cells and transformed cells were used to express the protein according to the manufacturer's instructions.
[0122] Other yeast vectors suitable for expressing IFNτ for use with methods of the present invention include 2 micron plasmid vectors (Ludwig, et al., 1993), yeast integrating plasmids (YIps; e.g., Shaw, et al., 1988), YEP vectors (Shen, et al., 1986), yeast centromere plasmids (YCps; e.g., Ernst, 1986), and other vectors with regulatable expression (Hitzeman, et al., 1988; Rutter, et al., 1988; Oeda, et al., 1988). Preferably, the vectors include an expression cassette containing an effective yeast promoter, such as the MFα1 promoter (Ernst, 1986; Bayne, et al., 1988, GADPH promoter (glyceraldehyde-3-phosphate-dehydrogenase; Wu, et al., 1991) or the galactose-inducible GAL10 promoter (Ludwig, et al., 1993; Feher, et al., 1989; Shen, et al., 1986). The yeast transformation host is typically Saccharomyces cerevisiae, however, as illustrated above, other yeast suitable for transformation can be used as well (e.g., Schizosaccharomyces pombe, Pichia pastoris and the like).
[0123] Further, a DNA encoding an IFNτ polypeptide can be cloned into any number of commercially available vectors to generate expression of the polypeptide in the appropriate host system. These systems include the above described bacterial and yeast expression systems as well as the following: baculovirus expression (Reilly, et al., 1992; Beames, et al., 1991; Clontech, Palo Alto Calif.); plant cell expression, transgenic plant expression (e.g., Gelvin and Schilperoot, 1988), and expression in mammalian cells (Clontech, Palo Alto Calif.; Gibco-BRL, Gaithersburg Md.). These recombinant polypeptides can be expressed as fusion proteins or as native proteins. A number of features can be engineered into the expression vectors, such as leader sequences which promote the secretion of the expressed sequences into culture medium. The recombinantly produced polypeptides are typically isolated from lysed cells or culture media. Purification can be carried out by methods known in the art including salt fractionation, ion exchange chromatography, and affinity chromatography. Immunoaffinity chromatography can be employed, as described above, using antibodies generated based on the IFNτ polypeptides.
[0124] In addition to recombinant methods, IFNτ proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies. Further, IFNτ peptides may be chemically synthesized using methods known to those skilled in the art.
[0125] III. Effectiveness of Orally-Administered IFNτ
[0126] Experiments performed in support of the present invention and detailed below demonstrate that orally-administered IFNτ polypeptide compositions are comparable in efficacy to injected IFNτ compositions with respect to the treatment of diseases or disease conditions which benefit from treatment with IFNτ.
[0127] Not only was orally-administered IFNτ effective at treating a disease benefiting from IFNτ treatment (EAE), but the oral route of administration resulted in unexpected advantages relative to treatment with injected IFNτ compositions. For example, orally-administered IFNτ resulted in a significantly lower level of anti-IFNτ antibodies in the serum of treated individuals (see Example 7). This is beneficial because the orally-administered IFNτ is therefore less likely to be rendered ineffective by a host immune response (i.e., desensitization to the treatment and/or dose level is significantly decreased), and the individual receiving the treatment is less likely to suffer adverse side effects as a result of such an immune response.
[0128] Results of experiments demonstrating these and related findings are presented below.
[0129] A. Orally-Administered TFNτ Inhibits Development of EAE
[0130] The efficacy of IFNτ in treating autoimmune disorders may be evaluated in rodents with experimental allergic encephalomyelitis (EAE; Zamvil and Steinman, 1990), an animal model of antigen-induced autoimmunity. EAE resembles human multiple sclerosis (MS) both in its clinical and pathological manifestations and can thus be used to assess treatments for human autoimmune diseases such as MS. EAE is a T-cell-mediated inflammatory autoimmune demyelinating disease induced by immunizing susceptible mouse, rat or guinea pig strains with myelin basic protein (MBP) or with encephalitogenic peptide fragments. Genetic susceptibility in the model animal strains is based in part on the capacity of encephalitogenic peptides to bind to particular class II major histocompatibility complex (MHC-II) molecules (Fritz, et al., 1983; Wraith, et al., 1989). In particular, mice having the H-2u haplotype are susceptible to EAE. Susceptible mouse strains include PL/J mice (Klein, et al., 1983), (PL/J×SJL)F1 mice (Zamvil and Steinman, 1990; Wraith, et al., 1989), B10.PL mice (Figuero, et al., 1982), NZW mice (Kotzin, et al., 1987), and (NZB×NZW)F1 (Kotzin, et al., 1987) mice.
[0131] Gamma-interferon (IFNγ) and beta-interferon (IFNβ) have been demonstrated to be effective in treating multiple sclerosis (Johnson, et al., 1994; IFNβ Multiple Sclerosis Study Group, 1993). In fact, IFNβ has been approved by the FDA as a therapeutic for multiple sclerosis. Although β-IFN is effective against MS, it has relatively high toxicity, and as a result, has a variety of undesirable side effects. As described herein, however, IFNτ has significantly lower toxicity that other interferons and may therefore exhibit fewer undesirable side effects.
[0132] In experiments performed in support of the present invention and detailed in Example 1, orally-administered and injected IFN-τ was tested for its ability to prevent the induction of EAE. EAE was induced in New Zealand White (NZW) mice by immunization with bovine myelin basic protein (bMBP). Recipient NZW mice received OvIFNτ by either i.p. injection or oral feeding 48 hours prior to, on the day of, and 48 hours after immunization with bovine myelin basic protein (bMBP) for induction of experimental allergic encephalomyelitis (EAE).
[0133] Both oral feeding and i.p. injection of OvIFNτ protected against EAE (Example 1, Table 3). All animals that received IFNτ via i.p. injection, and 7 of 9 animals that received IFNτ orally, were protected from symptoms of EAE. Furthermore, anti-OvIFNτ monoclonal antibody HL127 was effective at partially neutralizing the ability of the OvIFNτ to block EAE. These experiments demonstrate that orally-administered IFNτ is effective in treating symptoms of EAE, an animal model of multiple sclerosis.
[0134] B. OvIFNτ is Present in Sera Following Oral Administration
[0135] To confirm that orally-administered IFNτ enters the circulation, the sera of mice that received IFNτ by i.p injection or by oral administration were tested for the presence of IFNτ using a cytopathic effect (antiviral) assay (Familetti, et al., 1981) as described in Example 2.
[0136] The results are shown in FIG. 1. Specific activities are expressed in antiviral units/mg protein obtained from antiviral assays using MDBK cells. OvIFNτ was detected for up to two 2 hours following oral feeding (filled bars) at levels of 200 U/ml. These data indicate that orally-administered IFNτ enters the circulation and remains in serum for about two hours after being administered.
[0137] C. Lack of Toxicity from Orally-administered OvIFNτ
[0138] It has been previously demonstrated that the type I IFNs IFNα and IFNβ induced toxic side effects manifested as flu like symptoms, fever, nausea and malaise when used as therapeutics in humans (Degre, 1974; Fent and Zbinden, 1987). In contrast, OvIFNτ exhibits a remarkable lack of toxicity both in vitro and in vivo. Experiments performed in support of the present invention compared OvIFNτ with IFNs α and β for induction of toxicity as measured by lymphocyte depression in peripheral blood when given via oral feeding. Blood was obtained from the tail and white blood cells (WBC) counts were enumerated using a hemocytometer. Differential WBC counts were performed on Wright-Giemsa-stained blood smears.
[0139] The results are shown in Tables 2a, 2b and 2c, below. Significant levels of toxicity were detected in mice fed either IFN α and β while no significant lymphocyte depression was detected in mice fed 105, 2×105 or 5×105 U of OvIFNτ or PBS alone. These data suggest that orally-administered OvIFNτ has significantly-reduced toxicity with respect to other type I IFNS.
[0142] D. OvIFNτ Prevents Chronic Relapse of EAE
[0143] In addition to preventing the onset of symptoms associated with EAE, orally-administered OvIFNτ prevents paralysis in a chronic-relapsing model of EAE, as detailed in Example 3. Whereas 5/5 mice immunized with MBP (to induce EAE) which did not receive OvIFNτ treatment developed chronic relapsing paralysis, 4/5 animals treated with OvIFNτ (either i.p. injection or oral feeding, administered every 48 hours) were fully protected from the disease (FIGS. 2B and 2C). These data further support the results described above, and indicate that oral administration of IFNτ can block the development of chronic relapsing EAE. The experiments also suggest that orally-administration of IFNI as infrequently as once every 48 hours, over an extended period of time, is as effective as i.p. injection at treating a disease condition responsive to treatment by interferon-tau.
[0144] E. Histological Analyses of Spinal Chord from EAR Mice following Oral Administration of IFNτ
[0145] The ability of OvIFNτ to prevent EAE was also assayed by analyzing the effect of OvIFNτ treatment on cellular consequences of the disease, manifested in the central nervous system (CNS) as lymphocytic lesions in spinal cord white matter. The lesions are indicative of the extent of lymphocyte infiltration into the CNS. MBP-immunized mice were either not treated (control) or treated with OvIFNτ by oral or i.p. routes, and sections of the spinal cord lumbar region were stained and evaluated for lymphocytes as described in Example 4. Lymphocytic lesions were present in spinal cord white matter of control animals (FIG. 3A), but not in mice treated with OvIFNτ by i.p. injection (FIG. 3B) or oral feeding (FIG. 3C). These data indicate that the protective effect of IFNτ is associated with inhibition of lymphocyte infiltration of the CNS. Further, the data demonstrate that IFNτ treatment inhibits cellular manifestation of the autoimmune disease, rather than simply masking symptoms.
[0146] F. Cessation of Treatment with OvIFNτ Results in Relapsing Paralysis
[0147] Experiments detailed in Example 6 were performed to determine the type and duration of treatment effective to prevent EAE in mice injected with MBP. The mice were protected from EAE by OvIFNτ treatment via i.p. injection or oral feeding (every 48 hours) as long as the treatment persisted (58 days in Example 6), but developed symptoms of the disease after OvIFNτ treatment was stopped (FIG. 5). These results suggest that while IFNT may not cure an autoimmune condition like EAE (e.g., MS), it is an effective treatment that inhibits the pathological manifestations of the condition so long as treatment is continued.
[0148] G. Oral Administration of OvIFNτ Reduces Anti-OvIFNτ Antibody Response
[0149] As detailed in Example 7, one advantage of orally-administered (as opposed to injected) IFNτ treatment is a reduction in the anti-IFNτ antibody titer in individuals receiving the oral treatment. After removal of OvIFNτ treatment, mice from each treatment group were bled and sera were examined for the presence of anti-OvIFNτ antibodies by ELISA. Whereas mice receiving IFNτ by i.p. injection exhibited elevated levels of anti-IFNτ antibodies, animals receiving IFNτ by oral feeding exhibited much lower anti-IFNτ antibody titers (typically 3 to 5-fold lower). As expected mice which received no OvIFNτ treatment displayed no anti-OvIFNτ antibodies.
[0150] The sera were also examined for their ability to neutralize OvIFNτ antiviral activity on the MDBK cell line. None of the sera from either i.p. injected or orally fed mice possessed neutralizing activity (Table 4). These results suggest that oral feeding of OvIFNτ largely circumvents an antibody response directed against the OvIFNτ protein. Such a reduced antibody response in orally-treated subjects reduces the chance of undesirable immune system-related side effects of IFNτ treatment.
[0151] IV. Applications
[0152] A. IFNτ as a Treatment for Immune System Disorders
[0153] Diseases which may be treated using methods of the present invention include autoimmune, inflammatory, proliferative and hyperproliferative diseases, as well as cutaneous manifestations of immunologically mediated diseases. In particular, methods of the present invention are advantageous for treating conditions relating to immune system hypersensitivity. There are four types of immune system hypersensitivity (Clayman, 1991). Type I, or immediate/anaphylactic hypersensitivity, is due to mast cell degranulation in response to an allergen (e.g., pollen), and includes asthma, allergic rhinitis (hay fever), urticaria (hives), anaphylactic shock, and other illnesses of an allergic nature. Type II, or autoimmune hypersensitivity, is due to antibodies that are directed against perceived "antigens" on the body's own cells. Type III hypersensitivity is due to the formation of antigen/antibody immune complexes which lodge in various tissues and activate further immune responses, and is responsible for conditions such as serum sickness, allergic alveolitis, and the large swellings that sometimes form after booster vaccinations. Type IV hypersensitivity is due to the release of lymphokines from sensitized T-cells, which results in an inflammatory reaction. Examples include contact dermatitis, the rash of measles, and "allergic" reactions to certain drugs.
[0154] The mechanisms by which certain conditions may result in hypersensitivity in some individuals are generally not well understood, but may involve both genetic and extrinsic factors. For example, bacteria, viruses or drugs may play a role in triggering an autoimmune response in an individual who already has a genetic predisposition to the autoimmune disorder. It has been suggested that the incidence of some types of hypersensitivity may be correlated with others. For example, it has been proposed that individuals with certain common allergies are more susceptible to autoimmune disorders.
[0155] Autoimmune disorders may be loosely grouped into those primarily restricted to specific organs or tissues and those that affect the entire body. Examples of organ-specific disorders (with the organ affected) include multiple sclerosis (myelin coating on nerve processes), type I diabetes mellitus (pancreas), Hashimotos thyroiditis (thyroid gland), pernicious anemia (stomach), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junction), rheumatoid arthritis (joint lining), uveitis (eye), psoriasis (skin), Guillain-Barré Syndrome (nerve cells) and Grave's disease (thyroid). Systemic autoimmune diseases include systemic lupus erythematosus and dermatomyositis.
[0156] Other examples of hypersensitivity disorders include asthma, eczema, atopical dermatitis, contact dermatitis, other eczematous dermatitides, seborrheic dermatitis, rhinitis, Lichen planus, Pemplugus, bullous Pemphigoid, Epidermolysis bullosa, uritcaris, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Alopecia areata, atherosclerosis, primary biliary cirrhosis and nephrotic syndrome. Related diseases include intestinal inflammations, such as Coeliac disease, proctitis, eosinophilia gastroenteritis, mastocytosis, inflammatory bowel disease, Chrohn's disease and ulcerative colitis, as well as food-related allergies.
[0157] Autoimmune diseases particularly amenable for treatment using the methods of the present invention include multiple sclerosis, type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, uveitis, allergies and psoriasis.
[0158] Methods of the present invention may be used to therapeutically treat and thereby alleviate autoimmune disorders such as those discussed above. These treatments are exemplified herein with respect to the treatment of EAE, an animal model for multiple sclerosis.
[0159] B. IFNτ as Treatment for Reproductive Disorders
[0160] Although IFNτ bears some similarity to the IFNα family based on structure and its potent antiviral properties, the IFNαs do not possess the reproductive properties associated with IFNτ. For example, recombinant human IFNα had no effect on interestrous interval compared to IFNτ, even when administered at twice the dose (Davis, et al., 1992).
[0161] Therefore, although IFNτ has some structural similarities to other interferons, it has very distinctive properties of its own: for example, the capability of significantly influencing the biochemical events of the estrous cycle.
[0162] The IFNτ compositions of the present invention can be used in methods of enhancing fertility and prolonging the life span of the corpus luteum in female mammals as generally described in Hansen, et al. (1991), herein incorporated by reference. According to the teachings herein, such methods of enhancing fertility include oral administration of IFNτ in a therapeutically-effective amount. Further, the compositions may be similarly employed to regulate growth and development of uterine and/or fetal-placental tissues. Compositions containing human IFNτ are particularly useful for treatment of humans, since potential antigenic responses are less likely using a same-species protein.
[0163] C. IFNτ as an Antiviral Treatment
[0164] The antiviral activity of IFNτ has broad therapeutic applications without the toxic effects that are usually associated with IFNαs. As described above, IFNτ exerts its therapeutic activity without adverse effects on the cells. The relative lack of cytotoxicity of IFNτ makes it extremely valuable as an in vivo therapeutic agent and sets IFNτ apart from most other known antiviral agents and all other known interferons.
[0165] Formulations containing IFNτ can be orally-administered to inhibit viral replication. Further, the compositions can be employed in methods for affecting the immune relationship between fetus and mother, for example, in preventing transmission of maternal viruses (e.g., HIV) to the developing fetus. Compositions containing a human interferon-τ are particularly useful for treatment of humans, since potential antigenic responses are less likely using a homologous protein.
[0166] Examples of specific viral diseases which may be treated by orally-administered IFNτ include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, non-A, non-B, non-C hepatitis, Epstein-Barr viral infection, HIV infection, herpes virus (EB, CML, herpes simplex), papilloma, poxvirus, picorna virus, adeno virus, rhino virus, HTLV I, HTLV II, and human rotavirus.
[0167] D. IFNτ as an Antiproliferative Treatment
[0168] IFNτ exhibits potent anticellular proliferation activity. Accordingly, pharmaceutical compositions containing IFNτ, suitable for oral administration, can be used to inhibit cellular growth without the negative side effects associated with other interferons which are currently known. Such compositions or formulations can be used to inhibit, prevent, or slow tumor growth.
[0169] Examples of specific cell proliferation disorders which may be treated by orally-administered IFNτ include, but are not limited to, hairy cell leukemia, Kaposi's Sarcoma, chronic myelogenous leukemia, multiple myeloma, superficial bladder cancer, skin cancer (basal cell carcinoma and malignant melanoma), renal cell carcinoma, ovarian cancer, low grade lymphocytic and cutaneous T cell lymphoma, and glioma.
[0170] Furthermore, the development of certain tumors is mediated by estrogen. Experiments performed in support of the present invention indicate that IFNτ can suppress estrogen receptor numbers. Therefore, the IFNτ -containing compositions may be particularly useful in the treatment or prevention of estrogen-dependent tumors.
[0171] E. Veterinary Applications
[0172] In addition to the uses of the methods of the present invention detailed above, it will be appreciated that the methods may be applied to the treatment of a variety of immune system disorders suffered by domesticated and wild animals. For example, hypothyroidism in dogs typically results from a progressive destruction of the thyroid, which may be associated with Lymphocytic thyroiditis (Kemppainen and Clark, 1994). Lymphocytic thyroiditis, which resembles Hashimoto's thyroiditis in humans, is thought to be an autoimmune disorder. According to the guidance presented herein, hypothyroidism due to Lymphocytic thyroiditis in dogs may be treated with IFNτ as described above.
[0173] Another type of autoimmune disorder in dogs that may be alleviated by treatment with IFNτ is characterized by antinuclear antibody (ANA) positivity, pyrexia and seronegative arthritis (Day, et al., 1985). Immune-mediated thrombocytopenia (ITP; Kristensen, et al., 1994; Werner, et al., 1985), systemic lupus erythematosus (Kristensen, et al., 1994), and leukopenia and Coomb's positive hemolytic anemia (Werner, et al., 1985), may also be amenable to treatment using methods of the present invention.
[0174] V. IFN Pharmaceutical Composition Useful for Oral Administration
[0175] A. Formulation
[0176] Therapeutic preparations containing IFNτ or related polypeptides or proteins can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations comprising polypeptides like interferons have been previously described (e.g., Martin, 1976). In general, the IFNτ therapeutic compositions are formulated such that an effective amount of the IFNτ is combined with a suitable additive, carrier and/or excipient in order to facilitate effective oral administration of the composition. For example, tablets and capsules containing IFNτ may be prepared by combining IFNτ (e.g., lyophilized IFNτ protein) with additives such as pharmaceutically acceptable carriers (e.g., lactose, corn starch, light silicic anhydride, microcrystalline cellulose, sucrose), binders (e.g., alpha-form starch, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone), disintegrating agents (e.g., carboxymethylcellulose calcium, starch, low substituted hydroxy-propylcellulose), surfactants (e.g., Tween 80, polyoxyethylene-polyoxypropylene copolymer), antioxidants (e.g., L-cysteine, sodium sulfite, sodium ascorbate), lubricants (e.g., magnesium stearate, talc), or the like.
[0177] Further, IFNτ polypeptides of the present invention can be mixed with a solid, pulverulent or other carrier, for example lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, millopectine, cellulose derivative or gelatine, and may also include lubricants, such as magnesium or calcium stearate, or polyethylene glycol waxes compressed to the formation of tablets. By using several layers of the carrier or diluent, tablets operating with slow release can be prepared.
[0178] Liquid preparations for oral administration can be made in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1% to about 30% by weight of IFNτ, sugar and a mixture of ethanol, water, glycerol, propylene, glycol and possibly other additives of a conventional nature.
[0179] B. Dosage
[0180] An orally-active IFNT pharmaceutical composition is administered in a therapeutically-effective amount to an individual in need of treatment. The dose may vary considerably and is dependent on factors such as the seriousness of the disorder, the age and the weight of the patient, other medications that the patient may be taking and the like. This amount or dosage is typically determined by the attending physician. The dosage will typically be between about 1×105 and 1×108 units/day, preferably between about 1×106 and 1×107 units/day. It will be appreciated that because of its lower toxicity, IFNτ can be administered at higher doses than, for example, IFNβ. By way of comparison, patients with multiple sclerosis (MS) were treated with 106 U and 8×106 U of IFNβ. Patients receiving 8×106 U suffered fewer relapses of disease than did patients receiving 106 U. However, patients receiving the higher dose of IFNβ (8×106 U) also exhibited more side-effects associated with IFNβ's toxicity. In view of the lower toxicity of IFNτ, these higher effective dosages could be administered without the associated toxic side-effects.
[0181] Disorders requiring a steady elevated level of IFNτ in plasma will benefit from administration as often as about every two to four hours, while other disorders, such as MS, may be effectively treated by administering a therapeutically-effective dose at less frequent intervals, e.g., once every 48 hours. The rate of administration of individual doses is typically adjusted by an attending physician to enable administration of the lowest total dosage while alleviating the severity of the disease being treated.
[0182] Once improvement of a patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained.
[0183] C. Combination Therapies
[0184] It will, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies. For example, in view of IFNτ's relative lack of toxicity at high dosages, MS patients that do not show improvement at IFNβ1b's low dosage or could not tolerate IFNβ1b due to toxicity may benefit from subsequent or simultaneous treatment with higher dosages of IFNτ or peptides derived therefrom. Further, development of neutralizing antibodies has been demonstrated in IFNβ1b treated patients (Weinstock-Guttman, et al., 1995). In cases where such neutralizing antibodies prove to impede the effectiveness of IFNβ1b, IFNτ may be an important alternative therapy, since antibody cross-reactivity is unlikely to occur, and IFNτ is unlikely to generate neutralizing antibodies (see Example 7). Orally-administered IFNτ is particularly advantageous in this respect, since it causes a significantly lower anti-IFNτ antibody response than injected IFNτ.
[0185] Another type of combination therapy enabled by the present invention is the oral administration of an antigen against which an autoimmune response is directed in combination with IFNτ. Oral administration of such an antigen can result in tolerization, reducing the severity of the autoimmune disease (for review, see, e.g., Weiner, et al., 1994). It is contemplated that the IFNτ has a synergistic effect with the tolerization induced by the antigen, thereby alleviating the severity of the autoimmune disease. For example, MBP has been shown to suppress EAE (Lider, et al., 1989). According to the methods of the present invention, MBP may be administered in combination with IFNτ to treat multiple sclerosis. Other examples include administration of IFNτ with collagen to treat rheumatoid arthritis, and with acetylcholine receptor polypeptides to treat myasthenia gravis.
[0186] Furthermore, IFNτ may be orally administered with known immunosuppressants, such as steroids, to treat autoimmune diseases such a multiple sclerosis. The immunosuppressants may act synergistically with IFNτ and result in a more effective treatment that could be obtained with an equivalent dose of IFNτ or the immunosuppressant alone.
[0187] Similarly, in a treatment for a cancer or viral disease, IFNT may be administered in conjunction with, e.g., a therapeutically effective amount of one or more chemotherapy agents such as busulfan, 5-fluoro-uracil (5-FU), zidovudine (AZT), leucovorin, melphalan, prednisone, cyclophosphamide, dacarbazine, cisplatin, and dipyridamole.
[0188] The following examples illustrate but in no way are intended to limit the present invention.
[0189] VI. Materials and Methods
[0190] A. Buffers
[0191] Phosphate-buffered saline (PBS)
[0192] 10×stock solution, 1 liter:
[0193] 80 g NaCl
[0194] 2 g KCl
[0195] 11.5 g Na2HPO4-7H2O
[0196] 2 g KH2PO4
[0197] Working solution, pH 7.3:
[0198] 137 mM NaCl
[0199] 2.7 mM KCl
[0200] 4.3 mM Na2HPO4-7H2O
[0201] 1.4 mM KH2PO4
[0202] B. General ELISA Protocol for Detection of Antibodies
[0203] Polystyrene 96 well plates Immulon II (PGC) were coated with 5 μg/mL (100 μL per well) antigen in 0.1 M carbonate/bicarbonate buffer, pH 9.5. The plates were sealed with parafilm and stored at 4° C. overnight.
[0204] Following incubation, the plates were aspirated and blocked with 300 μL 10% NGS and incubated at 37° C. for 1 hr. The plates were then washed 5 times with PBS 0.5% "TWEEN-20". Antisera were diluted in 0.1 M PBS, pH 7.2. The desired dilution(s) of antisera (0.1 mL) were added to each well and the plates incubated 1 hour at 37° C. The plates were then washed 5 times with PBS 0.5% "TWEEN-20".
[0205] Horseradish peroxidase (HRP) conjugated goat anti-human antiserum (Cappel, Durham, N.C.) was diluted 1/5,000 in PBS. 0.1 mL of this solution was added to each well. The plate was incubated 30 min at 37° C., then washed 5 times with PBS.
[0206] Sigma ABTS (substrate) was prepared just prior to addition to the plate. The reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078 mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate was added to each well, then incubated for 30 min at room temperature. The reaction was stopped with the addition of 0.050 mL 5% SDS (w/v). The relative absorbance is determined at 410 nm.
[0207] C. Production of OvIFN-τ
[0208] A synthetic OvIFNτ gene was generated using standard molecular methods (Ausubel, et al., 1988) by ligating oligonucloetides containing contiguous portions of a DNA sequence encoding the OvIFNτ amino acid sequence (Imakawa, et al., 1987). The resulting IFNτ polynucleotide coding sequence spans position 16 through 531: a coding sequence of 172 amino acids.
[0209] The full length synthetic gene StuI/SstI fragment (540 bp) was cloned into a modified pIN III omp-A expression vector and transformed into a competent SB221 strain of E. coli. For expression of the IFNτ protein, cells carrying the expression vector were grown in L-broth containing ampicillin to an OD (550 nm) of 0.1-1, induced with IPTG (isopropyl-1-thio-b-D-galactoside) for 3 hours and harvested by centrifugation. Soluble recombinant IFNτ was liberated from the cells by sonication or osmotic fractionation.
[0210] For expression in yeast, the IFNτ gene was amplified using polymerase chain reaction (PCR; Mullis, 1987; Mullis, et al., 1987) with PCR primers containing Stul and Sacd restriction sites at the 5′ and 3′ ends, respectively. The amplified fragments were digested with StuI and SacII and ligated into the SacII and SmaI sites of "pBLUESCRIPT+(KS)", generating pBSY-IFNτ.
[0211] Plasmid pBSY-IFNτ was digested with SacII and EcoRV and the fragment containing the synthetic IFNτ gene was isolated. The yeast expression vector pBS24Ub (Sabin, et al., 1989; Ecker, et al., 1989) was digested with SalI. Blunt ends were generated using T4 DNA polymerase. The vector DNA was extracted with phenol and ethanol precipitated (Sambrook, et al., 1989). The recovered plasmid was digested with SacII, purified by agarose gel electrophoresis, and ligated to the SacII-EcoRV fragment isolated from pBSY-IFNτ. The resulting recombinant plasmid was designated pBS24Ub-IFNτ.
[0212] The recombinant plasmid pBS24Ub-IFNτ was transformed into E. coli. Recombinant clones containing the IFNτ insert were isolated and identified by restriction enzyme analysis. IFNτ coding sequences were isolated form pBS24Ub-IFNτ and cloned into a Pichia pastoris expression vector containing the alcohol oxidase (AOX1) promoter (Invitrogen, San Diego, Calif.). The vector was then used to transform Pichia pastoris GS115 His− host cells and protein was expressed following the manufacturer's instructions. The protein was secreted into the medium and purified by successive DEAE-cellulose and hydroxyapatite chromatography to electrophoretic homogeneity as determined by SDS-PAGE and silver staining. The purified protein had a specific activity of about 0.29 to about 0.44×108 U/mg as measured by anti-viral activity on Madin-Darby bovine kidney (MDBK) cells.
[0213] Orally-administered and injected IFN-τ was tested for its ability to prevent the induction of EAE. Recipient New Zealand White (NZW) mice received OvIFNτ (105 U/ml) by either i.p. injection or oral feeding 48 hours prior to, on the day of, and 48 hours after immunization with bovine myelin basic protein (bMBP) for induction of experimental allergic encephalomyelitis (EAE). 105 U of IFNτ were mixed with PBS to a total volume of 100 μl and administered using a feeding tube placed down the esophagus and into the stomach. The dilution of the IFNτ in PBS was done immediately before administration.
[0214] For induction of EAE in NZW mice, 300 μg of bovine myelin basic protein (bMBP) was emulsified in complete Freund's adjuvant (CFA) containing 8 mg/ml of H37Ra (Mycobacterium tuberculosis, Difco, Detroit, Mich.) and injected on either side of the base of the tail. On the day of immunization and 48 hours later, 400 ng of Pertussis toxin (List Biologicals, Campbell, Calif.) was also injected. For induction of EAE in SJL/J mice, the same protocol was used as described except mice were immunized again 7 days after the initial immunization. Mice were examined daily for signs of EAE and severity of disease was graded on the following scale: 1, loss of tail tone; 2, hind limb weakness; 3, paraparesis; 4, paraplegia; 5, moribund/death.
[0215] To determine whether prevention of EAE was specific to OvIFNτ treatment, an anti-OvIFNτ monoclonal antibody (mAb), HL127, was used to neutralize OvIFNτ ability to block EAE (antibody HL127, directed against aa 139-172 of SEQ ID NO: 2, neutralizes the antiviral activity of OvIFNτ in an antiviral assay using the MDBK cell line). A 1:10 dilution of HL127 was incubated for 2 hours with OvIFNτ prior to administration by either i.p. injection or oral feeding. Antibodies directed against IFNτ antigens, may be generated using the information herein combined with known techniques for antibody production (e.g., Harlow, et al., 1988).
[0216] The results are shown in Table 3, below. Both oral feeding and i.p. injection of OvIFNτ protected against acute induction of EAE. None of the animals that received IFNτ via i.p. injection developed symptoms of EAE, while of the animals that received IFNτ orally, 7 of 9 (78%) were protected. Anti-OvIFNτ antibody HL127 was effective at partially neutralizing the ability of the OvIFNτ to block EAE. These data indicate that orally-administered IFNτ is effective as a treatment in an animal model of multiple sclerosis.
[0217] The amount of OvIFNτ detectable in the sera of mice (treated as above) was compared over time after oral feeding or i.p. injection of OvIFNτ. Mice were administered 3×105 U of OvIFNτ and bled at 0.5, 2, 4, 6, 24 and 48 hours following IFNτ administration. Sera were tested in a cytopathic effect (viral plaque) assay (Familetti, et al., 1981) to determine the amount of IFNτ in the samples.
[0218] Briefly, dilutions of IFNτ were added to MDBK cells grown to confluency in a flat bottom 96 well plate and incubated for 18 to 24 hours at 37° C. Vesicular stomatatosis virus (VSV) was added to the plate for 45 minutes at room temperature. Virus was removed and methyl cellulose was added and the plate incubated for 48 hours at 37° C. After removal of methyl cellulose, the plate was stained with crystal violet for visualization of plaques. For measurement of IFN neutralization, OvIFNτ at a concentration of 500 U/ml was incubated for 1 hour at 37° C. with either sera or HL127 (a monoclonal specific of OvIFNτ). One antiviral unit caused a 50% reduction in destruction of the monolayer, relative to untreated MDBK cells infected with VSV (control plates). All samples were assayed simultaneously to eliminate interassay variability.
[0219] As shown in FIG. 1, OvIFNτ was detected at 0.5 hour and 2 hours after oral feeding (filled bars) at levels of 200 U/ml. By comparison, somewhat higher levels of OvIFNτ were detected for over a 24 hour period of time after i.p. injection (open bars). These data show that the above dose of IFNτ can be detected in serum for about two hours following oral administration.
[0220] The ability of OvIFNτ to prevent paralysis was examined using a chronic-relapsing model of EAE, in which SJL mice immunized with MBP develop a chronic form of the disease where the appearance of symptoms occurs in a relapsing-remitting manner (Zamvil and Steinman, 1990).
[0221] EAE was induced in SJL mice essentially as described above. The mice were treated with 105U of OvIFNτ by either i.p. injection or oral feeding on the day of immunization (day 0) and every 48 hours thereafter for the duration of the experiment. As presented in FIG. 2A, SJL mice which were immunized with MBP but did not receive OvIFNτ treatment developed chronic relapsing paralysis with a 5/5 incidence of disease, with a peak mean severity of ˜2.5 occurring 14 days after the start of the experiment. In contrast, treatment with OvIFNτ by either i.p. injection or oral feeding (FIGS. 2B and 2C, respectively) resulted in protection from EAE. Incidence of disease in both OvIFNτ treatment groups was reduced to 1/5 animals, with a mean severity of ˜1.0. These data indicate that oral administration of IFNT can block the development of chronic relapsing EAE, and suggest that orally-administered IFNτ may be as effective as i.p. injection when the IFNτ is fed about every 48 hours over an extended period of time.
[0222] Histological analyses were performed to determine the extent of lymphocyte infiltration into the CNS of MBP-immunized mice treated with OvIFNτ by oral and i.p. routes.
[0223] Mice were perfused with 4% paraformaldehyde, vertebral columns were removed and treated with formalin for 2 to 3 days. Spinal cords were dissected out and soaked in 0.5 % sucrose overnight at 4° C. Spinal cord sections were embedded and sections cut in a microtome. Sections were fixed to slides in 4% paraformaldehyde and stained with cresyl violet for visualization of inflammatory infiltrates.
[0224] The results are shown in FIGS. 3A, 3B and 3C at a final magnification of 222×. Lymphocytic lesions were present in control spinal cord white matter (FIG. 3A). In contrast, no lymphocytic infiltrates were detected in mice treated with OvIFNτ by i.p. injection (FIG. 3B) or oral feeding (FIG. 3C). These data suggest that the protective effect of IFNτ is associated with inhibition of lymphocyte infiltration of the CNS.
[0225] During the course of OvIFNτ treatment of SJL for prevention of chronic relapsing EAE, mice were bled and sera were examined for the presence of interleukin 10 (IL10). Sera from mice which received either a single IFNτ (105 U) treatment (by i.p. injection or oral feeding), prolonged IFNτ (105 U) treatment (by i.p. injection or oral treatment for greater than 20 days) or no treatment were examined for IL10 by enzyme-linked immunosorbent assay (ELISA) using IL10 ELISA kits (Genzyme, Cambridge, Mass.) following the manufacturer's instructions. All sera samples were tested in duplicate.
[0226] No IL10 was detected in control mice or in mice which received a single treatment of OvIFNτ by either i.p. injection or oral feeding. In contrast, SJL mice which received OvIFNτ by either i.p. injection or oral feeding every 48 hours for greater than 20 days had detectable levels of IL10 in their sera (FIG. 4). These data suggest that IFNτ-induced production of IL10 may be a contributing mechanism by which OvIFNτ prevents development of EAE.
[0227] SJL mice which were protected from EAE by OvIFNτ treatment via i.p. injection or oral feeding (every 48 hours) were followed for 58 days, during which time no disease development was observed. Treatment with OvIFNτ was then removed and the mice were observed for an additional 22 days for symptoms of disease.
[0228] The results are shown in FIG. 5. IFNτ treatment is denoted as plus signs and removal of IFNτ treatment is denoted as minus signs beneath the graph. Disease incidence in each treatment group was as follows: PBS control=3/4 (square); i.p. injection=3/3 (triangle); oral feeding=:3/4 (circle).
[0229] Both groups of mice which had previously been protected from EAE by OvIFNτ treatment developed signs of paralysis 6 to 12 days after removal of the OvIFNτ treatment. These data indicate that ongoing administration of IFNτ, by either i.p. injection or oral feeding, is desirable for continued protection from EAE in the chronic-relapsing model of EAE.
[0230] After removal of OvIFNτ treatment in the experiments described in Example 6, above, mice from each treatment group were bled and sera were examined for the presence of anti-OvIFNτ antibodies (Ab).
[0231] The antigen, OvIFNτ, was adsorbed to the flat bottoms of plastic tissue culture wells overnight at a concentration of 600 ng/well, and subsequently evaporated to dryness. The plates were treated with 5% milk (Carnation) in PBS for 2 hours in order to block nonspecific binding and then washed 3 times with PBS containing 0.05% Tween 20. Various dilutions of sera from mice which were untreated, OvIFNτ treated by i.p. injection and OvIFNτ treated by oral feeding were added and incubated for 3 hours. Binding was assessed with goat anti-mouse immunoglobulin coupled to horseradish peroxidase. Color development was monitored at 492 nm in an ELISA plate reader (Bio-Rad, Richmond, Calif.) after o-phenylenediamine and H2O2 were added and the reaction terminated with 2M H2SO4.
[0232] Exemplary results are shown in FIG. 6. Sera from untreated, OvIFNτ treated-i.p. injected and OvIFNτ treated-orally fed (2 mice/group) were examined by ELISA using multiple dilutions, including 1:30 (open bars) and 1:120 (filled bars). Mice which received OvIFNτ by oral feeding exhibited minimal Ab levels while mice which received OvIFNτ by i.p. injection exhibited elevated levels of anti-OvIFNτ Ab. As expected, mice which received no OvIFNτ treatment displayed no anti-OvIFNτ Ab.
[0233] Sera were also examined for their ability to neutralize OvIFNτ antiviral activity on MDBK cells as described above. The results are shown in Table 4, below. None of the sera from either i.p. injected or orally fed mice possessed neutralizing activity. These data suggest that oral treatment with IFNτ circumvents the Ab response directed against OvIFNτ protein observed in i.p. injection-treated individuals, and that neither treatment typically results in the generation of neutralizing antibodies.
[0234] While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.
Citas de patentes
Citada por
Eventos legales
Imagen original


text:
Tie Front Peplum Shirt
Style # 225880
Our versatile top features a slight peplum and has a ruched detail below the tie in front. Low V-neck and double button balloon sleeves. Style with sexy fitted pants.
- 100% Polyester
- Hand wash
- Imported
- Center back to hem: 21.75" (55 cm)
- Model is 5'10" and wears a US size S
Rated 4 out of 5�by 1 reviewer.
Rated 4 out of 5�by domesticdiva15 Sexy and classy This top is classy in a sexy way. Love the slits in the selves. It's one of those tops that will be timeless, which I love. True to size- 5'5 105lbs xs February 20, 2014
1-1 of 1


text:
In order to continue the campaign in Ottawa, Operation Red Nose Ottawa relies on:
By contributing as a sponsor, ORN Ottawa is enabled to help the community in the following ways:
If your organization is interested in supporting ORN Ottawa, please contact us to discuss the options further.
Specializes in vector graphics and web design. Providing web design, development and maintenance.
What Shopping Should Be. Providing Operation Red Nose Ottawa with office space for its basecamp.
RYTEC printing specializes in high volume digital copies, offset printing and graphic design. Generously printing all promotional materials (cards, posters, banners etc.)
Providing fast and reliable connections to homes and business since 1996. Official ORN Internet Service Provider.
A start-up entertainment company specializing in a wide spectrum of faculties like public relations, interactive digital media consultation, and event management and services. Providing communications - marketing consultation services
Bell is Canada's largest telecommunications company, providing Mobile phone, TV, high speed and wireless Internet, and residential Home phone services. Providing Smartphones and mobility data packages for road teams
Fast, warranty-approved oil changes and maintenance services. Mr. Lube provides quality car care services that extend the life of your vehicle. Providing oil change discounts for all volunteers and attendance draw prizes
Experience with all levels of government, domestic and international police, and security agencies. Provided cash donation
Since 1971, Starbucks Coffee Company has been committed to ethically sourcing and roasting the highest quality arabica coffee in the world.
Focussing on top quality, always fresh product, value, great service and community leadership,


text:
The 30-year-old suffered a setback on Monday after having recently returned to training following a knee injury.
Keller has confirmed that the striker will not feature for Schalke again this year.
"It doesn't really matter what the decision is on Klaas-Jan Huntelaar," Keller said.
"He can't play until Christmas. It doesn't matter if he decides to have an operation.
"He might recover without one. Both outcomes are possible. The decision has not been made yet.
"We didn't rush him back before he got injured. Usually an injury like his is fine after eight weeks. He felt good.
"He took part in everything and this setback was an accident. He collided with someone in a bad position. This would still have happened even if it was two weeks later."


text:
"SCISSORS, PAPER, ROCK is a book of insight and honest love; it has the lyricism of family legend, and the dead-on truthful ring of a report from the trenches. Fenton Johnson has made something wonderful here."
- Barbara Kingsolver, author of Pigs in Heaven
"The emotional power of SCISSORS, PAPER, ROCK resides on a plot of land in Kentucky…, [It] chronicles the courtships, the births, the deaths and the agonizing disappointments of two generations of the Hardins of Strang Knob…When we first meet Tom Hardin, the patriarch of his dwindling family, he is an old widower fighting terminal cancer and puttering away the last months of his life in his workshop. Unknown to Tom, his thirty-six-year-old son, Raphael, who has come to visit from his adopted home of San Francisco, is fighting battles of his own: not just AIDS, but anger, alienation, and an agonizing wistfulness…SCISSORS, PAPER, ROCK is a seductive rumination on the ways that memory can torment or soothe and sometimes do both at the same time."
- San Francisco Chronicle


text:
Four People Arrested After 6 Month Investigation
Four people are arrested after a six month investigation into the illegal distribution of Heroin and Crack Cocaine throughout the Greater Portland area.
28 year old Gabriel Williams, 20 year old Robert Combs and 23 year old Michael Fleury all of Brooklyn, NY along with 54 year old Denise Parham of Portland, Me were arrested and charged with Class B Unlawful Trafficking in Scheduled Drugs, Heroin and Crack Cocaine. Williams was also charged with Violating Conditions of Release as he was out on bail for a pending drug trafficking charge related to this investigation.
On October 7, 2013 at approximately 4:00 PM, agents and officers from the Portland Police Department executed a search warrant at 638 Congress Street. The search warrant was the result of an investigation that began approximately 7 months ago when residents reported to police a high volume of foot traffic and disturbances in the building. Agents observed a drug transaction take place between Parham and two other people. Utilizing this information along with information obtained over the past 6 months, agents obtained and executed a search warrant for apartment 309.
Evidence seized from the apartment included 72 grams of suspected Cocaine Base, 125 grams of suspected Heroin and $2773.00 dollars in suspected drug proceeds. The approximate street value of the seized drugs is $30,000 dollars. All four suspects were transported to the Cumberland County Jail where bail was set at $50,000 dollars each. The investigation is ongoing and other arrests are expected.
Category: News


text:
Bonasa umbellus
IUCN Conservation Status: Least Concern
Tim Lenz
The dappled, grayish or reddish Ruffed Grouse is hard to see, but its "drumming on air" display is a fixture of many spring forests. It can come as a surprise to learn this distant sound, like an engine trying to start, comes from a bird at all. This plump grouse has a cocky crest and a tail marked by a broad, dark band near the tip. Displaying males expose a rich black ruff of neck feathers, giving them their name.
Listen as Macaulay Library Audio Archivist Martha Fischer describes listening to the drum of a male Ruffed Grouse. Cornell Lab of Ornithology;
Play Clip
Seeing the secretive Ruffed Grouse can be quite difficult—although it can be easy to hear them when they are drumming. To track one down, note the locations where you hear drumming males—this is generally most frequent very early in the morning. Otherwise, you may encounter foraging birds simply by walking slowly and quietly through appropriate forest, or while driving along narrow forested roads. In winter, watch for Ruffed Grouse feeding on deciduous-tree buds in bare treetops along a road.
Contact the Lab | Privacy Policy | Terms of Use | Site Credits Copyright © 2015 Cornell University


text:
The Faculty of Arts and Sciences has announced that Earl C. Ravenal '52, of Providence, Rhode Island, and Eliot House, has been awarded the History and Literature Prize for 1950-51. The award is $50 for the purchase of any kind of book except current fiction.
Want to keep up with breaking news? Get the latest, straight to your inbox.


text:
The radical thing is that in the Old Testament, everybody came together and sang. And in the New Testament, the Jew and the Gentile, the Greek and the Roman, the young and the old all came together and sang together. That's the witness of church history. It's not some kind of food court where everyone chooses their favorite music and goes that direction.
Paul told persecuted churches to get together and teach and admonish one another and sing psalms, hymns, and spiritual songs. There's no intellectual reason to do that; there's no church- growth reason to do that. It's a command, because that is the radical thing.
Listen at the graceslo websiteListen at the itunes podcast
Business CentsSage Theule, a fifth-grader in California, started a club to change one little girl's life. This is her story:"I decided to sponsor a child just with girls from my school. I picked Yenifer because she was 3 and I wanted a girl who was younger so we could sponsor her for longer. "To earn money for sponsoring Yenifer, I started the Compassion Girls' Club. There are nine girls in the club. We make and sell earrings, bracelets and beaded bookmarks. Also we have a lemonade stand. I made a logo to put on everything we sell. It's a heart with wings on it to send out love. And I made handouts with Compassion's Web site on them. "I think my sponsorship means a lot to Yenifer. She always sounds really happy in her letters. Also from my sponsorship I learned that when you're helping others it makes you and them feel good."
"The Set-Apart Family" is the theme for the 2008 Family Camps. Through speakers, small groups, family exercises, and peer groups, we will look at the life of Moses as an example of being a person "set apart" for a purpose, a passion, a purity, and a partnership. Mornings are set aside for peer group activities and teaching for all ages. Adults meet in small groups for discussion of the topic of the day, followed by a presentation from our resource person or couple for the week. Afternoons are free for relaxing and enjoying recreation with family and friends. Evening activities vary from seminars on marriage and parenting to square dancing for the whole family. Through all these activities our goal is to become a more "set apart" family that honors God and each other and makes the gospel appealing to those we come in contact with.
via a reader
or enter your email address...


text:
EAGLES EXTRAS: Mack's the man?
I'm no Stephen A. Smith, but I've got sources, too.
Check back with me in a few hours, because N.C. Central is getting ready to name its next football coach.
Dwayne Foster was NCCU's interim coach during this past 5-7 season for the Eagles when he replaced Henry Frazier III.
NCCU athletics director Ingrid Wicker-McCree fired Frazier in August because headlines about his personal life were taking away the shine of the winning season he secured for the school in 2012. NCCU hadn't had a winning season since 2007.
Foster wants to stay on as NCCU's coach, but there's been no word out of the university as to whether he even was granted an interview.
There's been no official correspondence from NCCU about any of the coaching candidates.
But stuff leaks, and what's widely known is that former Winston-Salem State coach Connell Maynor was in the mix for the NCCU job, yet Hampton athletics director Novelle Dickenson announced on Tuesday that Maynor was his new football coach and off the market.
Still on the board for NCCU were Southern University coach Dawson Odums, University of South Alabama wide receivers coach Jerry Mack and Nevada University tight ends coach James Spady.
Now, I recently got off the phone with black-college sports insider Donald Ware, who'd recently gotten off the phone with Odums, who said he ain't going nowhere. Odums is staying at Southern.
In fact, Odums and Southern have agreed in principle to new contract terms.
Spady used to coach at NCCU, but it really looks as if Mack will be the guy Wicker-McCree brings to the microphone during the on-campus press conference in just a few.
-- John McCann| email@example.com| @johntmccann


text:
Show Me Your "Monster" Kits! (let's say... 7 piece and up)
View Single Post
Platinum Member
Join Date: Apr 2006
Location: Boise, Idaho
Posts: 7,236
Re: Show Me Your "Monster" Kits! (let's say... 7 piece and up)
Originally Posted by
This might not be a 'monster' kit amoung true 'monster' kits but for me it's big enough - I usually play a 4pc kit.
This is what my Purewood African Mahogany 6pc Gretsch kit evolved to a couple of years after I bought it. I bought it new in 2007 - one of only two shellpacks available to Canada. They had to be ordered/sent from the Factory in Taiwan. There were only 60 shellpacks made for world-wide sale. They are incredibley nice drums and I stack them up to Gretsch Custom USA any day....I can say that because I have a new Custom USA kit and three Vintage Gretsch kits.
The photo was taken in 2009 - I don't have the rack (Gibraltar DBL Bass Rack) I traded it for some new Gibraltar Jazz Series stands and I love them.
I added the Remo Roto toms that I've had since I bought them new in the early 70's - 12/10/8 and I added a 6 a couple of years ago. The lighter drum on the far right is a Gretsch Mighty Mini snare.
My "Little" Monster Kit............
That's a beautiful kit, but it's really not Monster. To me, that's 9 pieces and up. 5 to 8 is mid sized.
Ironwood kit
Tiki kit
View Public Profile
Send a private message to Deathmetalconga
Find all posts by Deathmetalconga


text: Basically on this thread, you will post videos of yourself supporting El Tri or things you've done. Forget the monetary things like paying for a ticket or buying a jersey. Post yourself wearing la verde or videos that you made whilst being on a match. Etc... or just your pals drinking some coronas. You know, we criticize the team a lot, they deserve a *little support. I'll start by posting this RANDOM video I found on the net, it was a game from two days ago or something. Not that I watched. [ame="http://www.youtube.com/watch?v=BlGYhu8r2JA"]YouTube - Mexico 5 vs USA 0 mexico gold cup champions (copa oro) HD televisa[/ame]


text:
Hanley teenager Nicholas Wilkinson is jailed after fleeing police
Posted: August 23, 2014|
TEENAGER Nicholas Wilkinson has been jailed after he gave police the slip.
The 19-year-old was stopped by officers who told him he was wanted for a recall to custody.
Stoke-on-Trent Crown Court was told that Wilkinson was co-operative to begin with but ran off as the officer reached for her handcuffs. Despite an area search carried out by the police he was not found.
Twelve days later officers saw Wilkinson in a block of flats in Hanley.
Related content
He ran off but was chased and caught. The court was told he resisted arrest and was abusive to the custody officer at the Northern Area Custody Facility.
Wlkinson, of Linfield Road, Hanley, pleaded guilty to escape on July 17 and resisting a police officer in the execution of his duty on July 29.
Yesterday Wilkinson was jailed for nine months.
Recorder Andrew Easteal branded his actions 'unpleasant and completely unnecessary.'
Sentencing him, Mr Easteal said: "You pushed the officers aside and ran from them. It was unlawful and stupid.
"On July 29 you behaved disgracefully. These police officers were doing their duty as they have to do.
"What you did was unpleasant, violent, abusive and completely unnecessary."


text:
Name: Lichtenstein
Location: Baden-Wurttemberg
Country: Germany
this castle
Perched on a rock high above the valley floor, a product of the
romantic passion for castles in the nineteenth century. It was built from 1840
to 1841 by Duke Wihelm of Urach.
Have you visited a castle?
Do you know something about a castle?
If so, write something about it here.
Review this castle
Discount Tours
Discount Cruises
Discount River Cruises
Globus Tours
Trafalgar Tours
Disney Vacations
Quinceaneras at Sea
Tauck Tours
CIE Tours
Cosmos Tours
Affordable Travel
Affordable Vacations
Celebrity Cruises
Holland America Cruises
Norwegian Cruises
Oceania Cruises
Princess Cruises
Royal Caribbean Cruises
Azamara Cruises
Crystal Cruises
Cunard Cruises
Disney Cruises
MSC Cruises
Regent Cruises
Seabourn Cruises
Silversea Cruises
Windstar Cruises
Avalon Waterways Cruises
Uniworld Cruises
Viking Cruises
Tauck Cruises


text:
The critics from the group of Human Rights, which once addressed that, at times it becomes difficult or rather unsafe to reveal the real identity. Considering this fact, the search engine giant GOOGLE, has amended a naming policy on its social network –> Google+.
Few hours back, Bradley Horowitz, The Vice President of Google+, posted an article naming "Toward a more inclusive naming policy for Google+
In his post he said that,
When we analyze the set of all name appeals on Google+, we find that they generally fall into three major categories:
– The majority (60%) of these users want to simply add nicknames.
– About 20% of appeals are actually businesses (who are inadvertently trying to set up their business as a Profile, rather than using Google+ Pages which were intended for this purpose.)
– And the remaining 20% would either prefer to use a pseudonym or another unconventional name.
Also at times, names in another script have been found on Google+. झैनील डेढिया (Zainil Dedhia written in an Indian Language). Further people use Zainil "The Rock Star".
Bradley wants to make Google+ like sharing in the real world. Thus, today in the naming policy, he announced that, the users of Google+ will be able to add alternate names on their Google+ profile.
But it should be noted that, once you change your name on the Google+ profile, the changed name will reflect on all the services of Google! Hence now with the new naming policy of Google+, you will be able to use the common name of yours on all the services of Google.
To change your name on Google+, all you need to do is, go to your Google+ profile. On the top right hand side, click on Edit Profile (Blue Button). As you click on that button, the page display will change. Now click on your name. Once you click on your name, a frame will open which will have a link "+ More option", click on it. Now add your new details & change your display name.
Google+ Profile -> Edit Profile -> Select Your Name -> More Options.
Also Bradley said that,
Today we're pleased to be launching features that will address and remedy the majority of these issues. To be clear – our work here isn't done, but I'm really pleased to be shipping a milestone on our journey.
And finally he concluded saying that,
Today is a small step towards improving the ways in which you can communicate your identity on Google+. We will be listening to feedback from the community and will continue to refine all aspects of how we handle names and identity over the coming weeks, months and beyond.
Conclusion: With the new naming policy of Google+, you can use pseudonyms in your profile name.