text:
Chronology of the Old Testament - eBook
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Product Description
The goal of this book is to demonstrate "that every chronological statement contained in the Sacred Writ is consistent with all other chronological statements contained there-in. "The author carefully and thoroughly investigates the chronological and mathematical facts of the Old Testament, proving them to be accurate and reliable. This biblically sound, scholarly, and easy-to-understand book will enlighten and astound its readers with solutions and alternatives to many questions Bible scholars have had over the centuries.
Were there 66, 70, or 75 "souls" in Egypt when Jacob arrived? Were the Hebrews in Egypt for 430 years, or a shorter length of time? How long did Jacob have to wait before marrying the first of Laban's daughters, and how long did he wait for the second? What year was Christ born? With reliable explanatory text, charts, and diagrams, this book provides a systematic framework of the chronology of the Bible from Genesis through the life of Christ. Features:
- Solution to the chronology of Judges
- Solution to Daniel 9:2, the 483-year prophecy
- Solution to the length of the Hebrews' sojourn in Egypt
- Solution to the "number of souls" in Egypt when Jacob came there
- Chronology of the life of Christ
- Chronology of the kings of the divided monarchy
- 48 charts, graphs, and diagrams
- Fully indexed/complete bibliography
Product Information
|Format: DRM Free ePub
Vendor: Master Books
Publication Date: 2005
Availability: In Stock
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Author Bio
Following a 14-year professional career during which he held varying positions of responsibility as Paleontologist, Geophysicist, District Geophysicist, Geophysical Manager, and Regional Geophysicist with Texaco and Tenneco respectively, Dr. Floyd Nolen Jones was selected to attend Division Manager School shortly before resigning from his scientific vocation in 1974 to pursue Biblical studies.
Having attained a Ph.D. as well as a Th.D., Dr. Jones has garnered majors in the disciplines of Geology, Chemistry, Mathematics, Theology, and Education from six institutions of higher learning. A magna cum laude graduate and an ex-evolutionist, he also possesses a minor in Physics and is an ordained Minister (SBC).
Dr. Jones twice served as adjunct Professor at Continental Bible College in Brussels, Belgium. Having authored a definitive work on bible chronology, he has also written several books on textual criticism in defense of the traditional biblical text. He is currently engaged in ongoing Biblical research and the teaching of God's infallible Word. He and his wife, as well as his children and grandchildren, reside in Texas.
Product Reviews
Declared Unto HimAge: 35-44Gender: female5 Stars Out Of 5So much infoJanuary 8, 2012Declared Unto HimAge: 35-44Gender: femaleQuality: 5Value: 4Meets Expectations: 5This review was written for The Chronology of the Old Testament--Book and CD-ROM.Honestly, this would be an invaluable resource for any bible student, scholar or home learning family. The Chronology of the Old Testament has over two hundred pages of intense, biblical chronology including various historical documentation, notations and charts.
A priceless companion to Ussher's Annal's of the World, but make the addition of Adams Map of History along with a biblically based history curriculum, and you would have a serious, solid foundation for history ~ perfect for all ages.


text:
By ZoomerThink you've been there, done Europe? Reignite your wanderlust with these six inspiring ways to experience the Old World anew. 1. The South of France The antithesis of a bucket list goes sort of like this for me: Rather than experiencing something once...Read More»
By Bert ArcherA dollar you save on things that don't pay you back in joy, delight or awe is a dollar you can redirect to things that do. Here, how to get the most for your travel buck. When it comes to travel, old habits can be expensive. For instance, I...Read More»
7 Common Household Items That Make Great Travel Gadgets
10 Amazing Places To Vacation With Your Dog
10 Romantic Destinations to Swoon Over
9 Facts You (Probably) Didn't Know About Brazil
By Laura Grande

subset: commoncrawl


text:
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text:
The maturation of epidermin requires a series of modification reactions to change its chemical structure. EpiD protein, whose three-dimensional structure is shown here, catalyzes the first modification of epidermin, the decarboxylation of the C-terminal cysteine residue. EpiD is essential for epidermin biosynthesis.
Information about structure of epidermin-biosynthesis protein EpiD can be found at the Center for Structural Genomics of Infectious Diseases (CSGID) Web page, and the Protein Data Bank entry 3QJG.
All featured structures from the NIAID Structural Genomics Centers
Last Updated April 01, 2011


text:
- Report: #728092
Report - Rebuttal - Arbitrate
Complaint Review: Hewlett-Packard
Hewlett-Packard3000 Hanover Street, MS 1050 Palo Alto, California United States of America
Hewlett-Packard HP, HP sold me a lemon laptop, told me to return it for a refund but failed to make the refund Palo Alto, California
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HP sold me a lemon computer, it completely died after 2 months of use. They were not able to repair it and I was told to send it back and I would receive a refund of $1,108.48. I sent it back and have proof of receipt month ago but HP has failed to refund my money, despite many emails and phone calls.
I purchased an HP laptop computer online via the HP Homestore, with my VISA card on 9/7/2010. It was shipped to my mailing address (my stepdaughter's home) in Oakland, as we live in Panama. (We live on a remote island and cannot receive mail there, all mail is sent to our Oakland mailing address, above). My husband was visiting there at the time and he brought it down with him.
The computer was put into use on or about 11/30//2010 and it worked until 1/30/2011, when it simply died. It could not be started. The cord and adapter worked fine when tested, the computer was simply dead. Tech support said there was a power problem with the motherboard and it would have to be returned for warranty repair.
HP refused to honor the Global Warranty I had purchased (and which they promote on their website) and required me to send the computer back to the USA for warranty repair, which I did.
When I received the computer back, it was as dead as when I sent it back to them. HP failed to repair it, instead the replaced the adapter (which was not the problem).
After over 50 phone calls and many emails to the "Case Management" Department and "Executive Customer Relations" Department, (all of which I can document) I finally was promised a refund and told to return the computer, which I did (and I have proof of receipt from Federal Express.) The refund promised was $1,108.48 (which is less than the amount I paid for the computer but I agreed to accept it.)
HP received the computer on April 11, 2011. It is now May 11, 2011. The "case manager" assigned to my case by HP claims that a check was sent via "regular" (first class) mail on April 12. But it was never received. If it was even sent at all, it cannot be traced because HP chose not to send it certified or express mail or by any trackable method. The Post Office informs me that they cannot track it for that reason. HP refused to send me any proof that the check was even sent, they claim they cannot do so because it was not sent by any trackable modality.
UPDATE: On May 9, I was told that the original check had been cancelled and a new check sent. My "case manager" agreed to send me tracking information but a day later told me that the check could not be tracked because it was sent 3rd or 4th class mail (ie, "junk mail") and it is against company policy to send refunds in any way that can be tracked. The second check has not been received, and I doubt that it will be, since it was not addressed as I had instructed. For the past 3 weeks, I have repeatedly requested that IF a check was sent it was not timely received and that payment be stopped and the funds instead be wired to my California credit union account or alternatively, that a new check be sent via some method that provides for tracking and proof of delivery (ie, express mail, federal express, or first class mail return receipt requested).
But my "case manager" tells me that it is against company policy to send refunds via any trackable method and the company has no obligation to do anything further. She said that the company is only obligated to "put a check in the mail" not to do so in a way that ensures it will be received and not lost, stolen, or sent to the dead letter box. In other words, I am not getting my refund.
In sum, I was promised that if I returned the computer, I would get a refund. I relied on that promise and returned the computer, HP received it, but did not refund my money. That's fraud.
I have filed consumer fraud complaints with the Federal Trade Commission and the California Attorney General's office and will also be filing a compliant with the Better Business Bureau. Because we live in Panama and have only a mailing address in California, it is financially prohibitive for us to return to file a small claims court action now, but we will do so whenever we next schedule a visit to California if HP continues to refuse to refund our money and if the statute of limitations hasn't expired by then.
This report was posted on Ripoff Report on 05/11/2011 12:39 PM and is a permanent record located here: http://www.ripoffreport.com/r/Hewlett-Packard/Palo-Alto-California-94304/Hewlett-Packard-HP-HP-sold-me-a-lemon-laptop-told-me-to-return-it-for-a-refund-but-faile-728092. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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text:
Cottage Lake
19150 NE Woodinville Duvall Rd
Woodinville, WA 98077 Phone number & directions
19150 NE Woodinville Duvall Rd
Woodinville, WA 98077 Phone number & directions
License & Identifications
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Joo-Won Yoon:
View the full profile to get information on:
- Qualifications
- What's important to you
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Home > Find a Dentist > WA > Woodinville Dentists > Dr. Joo-Won Yoon, DDS
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- Additional Information
- Dr. Joo-Won F Yoon DDS is a female dentist and practices in Family Dentistry.
- Where does Dr. Joo-Won F. Yoon practice?
- Dr. Joo-Won Yoon has 2 office locations.
Dr. Joo-Won Yoon practices Family Dentistry near Woodinville, WA.
Additional practice locations include: Seattle, WA.
See Woodinville Dentists.


text:
So, the Shockwaves NME Awards 2009 are well and truly over and the entire office (plus half of the rock community, several hundred Cure fans and Sarah Harding) are nursing biblical hangovers, including we suspect this guy:
(We haven't worked out the identity of the fool yet, just that he's not in a band and one passer-by labelled him an "industry douchebag").
Anyway, here's that winners list in full. Glad to see Muse win Best Live Band again? Pissed that Barack beat Brandon as Hero Of The Year? Let us know what you think below and let battle commence...
Best British Band
Winner - Oasis
Nominated - Bloc Party, Radiohead, Muse, Last Shadow Puppets
Best International Band
Winner - The Killers
Nominated - MGMT, Kings Of Leon, Vampire Weekend, Crystal Castles
Best Solo Artist
Winner - Peter Doherty
Nominated - Lightspeed Champion, Ladyhawke, Jay-Z, Laura Marling
Best New Band
Winner - MGMT
Nominated - Vampire Weekend, Glasvegas, Late Of The Pier, White Lies
Best Live Band
Winner - Muse
Nominated - Radiohead, Oasis, The Killers, Kings Of Leon
Best Album
Winner - Kings Of Leon ('Only By The Night')
Nominated - The Killers, Glasvegas, Oasis, Bloc Party
Best Track
Winner - MGMT ('Time To Pretend')
Nominated - Vampire Weekend ('A-Punk'), The Last Shadow Puppets ('The Age Of The Understatement'), Kings Of Leon ('Sex On Fire'), The Ting Tings ('That's Not My Name')
Best Video
Winner - The Last Shadow Puppets ('My Mistakes Were Made For You')
Nominated - Late Of The Pier ('Heartbeat'), Radiohead ('House Of Cards'), Vampire Weekend ('A-Punk'), Oasis ('The Shock Of The Lightning')
Best Live Event
Winner - Glastonbury
Nominated - T In The Park, Isle Of Wight Festival, Reading & Leeds Festivals, V Festival
Best TV Show
Winner - The Mighty Boosh
Nominated - Skins, Gavin & Stacey, Never Mind The Buzzcocks, The IT Crowd
Best Dancefloor Filler
Winner - Dizzee Rascal ('Dance Wiv Me')
Nominated - Crystal Castles ('Courtship Dating'), Friendly Fires ('Paris'), Bloc Party ('Mercury'), Late Of The Pier ('Bathroom Gurgle')
Best DVD
Winner - Arctic Monkeys At The Apollo
Nominated - Foo Fighters: 'Live At Wembley', The Rolling Stones: 'Shine A Light', Muse 'HAARP', Kaiser Chiefs: 'Live From Elland Road'
Best Band Blog
Winner - Noel Gallagher / Oasis
Nominated - Little Boots, Lightspeed Champion, Radiohead, Foals
Best Venue
Winner - London Astoria
Nominated - O2 Arena, Glasgow Barrowland, O2 Academy Brixton, Manchester Apollo
Best Album Artwork
Winner - Muse ('HAARP')
Nominated - We Are Scientists ('Brain Thrust Mastery'), The Cure ('4.13 Dream'), Guillemots ('Red'), The Killers ('Day & Age')
Hero Of The Year
Winner - Barack Obama
Nominated - Brandon Flowers, Noel Gallagher, Noel Fielding, Alex Turner
Villain Of The Year
Winner - George Bush
Nominated - Peter Doherty, Amy Winehouse, John McCain, Gordon Brown
Best Dressed
Winner - Alexa Chung
Nominated - Noel Fielding, Brandon Flowers, Alex Turner, Noel Gallagher
Worst Dressed
Winner - Amy Winehouse
Nominated - Katy Perry, Peter Doherty, Brandon Flowers, Johnny Borrell
Worst Album
Winner - Jonas Brothers ('A Little Bit Longer')
Nominated - Coldplay ('Viva La Vida Or Death And All His Friends'), Britney Spears ('Circus'), Scouting For Girls ('Scouting For Girls'), Razorlight ('Slipway Fires')
Worst Band
Winner - Jonas Brothers
Nominated - Tokio Hotel, Scouting For Girls, Fall Out Boy, Oasis
Sexiest Male
Winner - Matt Bellamy
Nominated - Carl Barat, Keith Murray, Peter Doherty, Miles Kane
Sexiest Female
Winner - Hayley Williams
Nominated - Kate Jackson, Alison Mosshart, Lykke Li, Stephanie Dohen
Best Website
Winner - YouTube
Nominated - Facebook, Last FM, MySpace, Bebo


text:
Sighting Basics
Sighting Location
|New York
|United States
Sighting Specifics
|Viewing Distance
|Over 1 Mile, Unknown
|Sighting Duration
|Object Features
|Object Flight Path
|Straight-line path
|Object Shape
|Weather Factors
Sighting Details
This is my grandfather's story, He was sitting in his recliner watching basketball on TV, he then noticed a small light out the bay window going in a straight path traveling north in the east sky over the hill across town. After a few moments he then witnessed three longer cigar shaped lights traveling the same line. He first thought was that the first light was a plane with its landing lights on that would circle around before heading into the county to the south, we see that from time to time. He thought this until he noticed the three larger cigar shaped lights that seemed to be following it. They were moving very slowly. What he thought was strange was that there weren't any blinking lights at all. He continued to watch these lights for about fifteen minutes and they got behind some pine trees in his viewing way. After that they disappeared. He assumes they went east and did it quickly. He isn't very rattled from the sighting just curious about what he saw.
Did You See It? Have a Comment or Question? Discuss it Below.
blog comments powered by Disqus
- There has been one other sighting in Marathon
- There were no other sightings in Marathon on or around February 22nd 2014
- This sighting has never been favorited.


text:
City Girl - eBook
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Product Description
Product Information
|Format: DRM Protected ePub
Vendor: Harvest House Publishers
Publication Date: 2008
Availability: In Stock
Series: Yellow Rose
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Publisher's Description
City girl Reagan Sullivan may know her way around New York City, but nothing back East has prepared her for the land of armadillos and tall Texans. Chasing after adventure to blur an unhappy past, the highly independent Reagan has landed a job out West-but quite clearly she is not out to land a husband. When the gentle rancher Cash Rawlings comes into her life, Reagan finds herself intrigued with the man and his faith-but love and commitment are adventures this city girl has vowed never to embark upon.
Will Reagan be able to come to terms with her fragile past and discover a new way of living without fear? And will the faith and peace that belong to so many of her new friends ever enter her own life?
About This Series: Grab your hat and horse and head to the Lone Star state in the pages of the popular Yellow Rose Trilogy (nearly 500,000 sold)! Lori's engaging characters, heartwarming romances, and inspirational truths team with fresh new covers to please fans and win new readers everywhere.
Product Reviews
Donna Andrade5 Stars Out Of 5July 18, 2010Donna AndradeGood ending to the Rawlings family series. Not only does each brother find love, but the whole family comes to know the truest Love of all.
Lisa Kramer5 Stars Out Of 5April 11, 2009Lisa KramerCity Girl is a good book as well. The way Reagan handles the men who try to ask her out is amazing. I've never seen that in any other book I've read, except in Pretense when Delancey made it known in no uncertain terms to Chet that she was a black belt. That was one of my most favorite parts of the book. My favorite parts of the book Pretense, however, were whenever Mackenzie and Delancey came to Christ after pushing him away for so many years. Anyway, back to the review for City Girl. I love it when Cash and Reagan get married. They are so in love it isn't even funny.
Yolanda Metcalf5 Stars Out Of 5July 23, 2008Yolanda MetcalfThis is a great reading book, I really enjoyed the story line, I would recomend this book to any one for lite reading,
Jessica H4 Stars Out Of 5June 5, 2008Jessica HLori Wick has been one of my favorite authors for a long time. I enjoyed this series by her. Cash is probably my favorite Rawlings in this series and it was a nice, light read to just relax with.


text:
|Publication number
|Publication type
|Application number
|Publication date
|Feb 18, 1986
|Filing date
|Jan 25, 1985
|Priority date
|Aug 25, 1983
|Fee status
|Publication number
|John C. Houck, Leonard M. Patt
|Original Assignee
|Endorphin, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (4), Non-Patent Citations (32), Referenced by (66), Classifications (19), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 526,356, filed Aug. 25, 1983, now abandoned, which was a continuation-in-part application of Ser. No. 419,995, filed Sept. 20, 1982, now abandoned.
Mammals and birds, particularly commercial poultry flocks, are often at risk due to exposure to various viral or fungal infections. Such exposure can arise in the natural environment, particularly the dairy or poultry farm, or from unusual situations such as surgery, accidents or wounds. The mammalian body defends against viral and fungal infections by an immunological sequence of events stimulated by the presence of the invasive entity such as virus, bacteria or fungus, the end result of which is the production of T-cells which act in conjunction with macrophages to promote phagocytosis.
Some mammals are deficient in this immunological response either because of some metabolic malfunction, or due to medical treatment such as chemotherapy or spleenectomy. Many domestic animals, particularly poultry, seem to be constantly susceptible to such infections and do not respond immunologically with sufficient rapidity to prevent the loss of large numbers of them. Turkey flocks are especially sensitive in this regard. This leads to significant increases in the costs of turkey husbandry.
A material to enhance the immunological response of mammals to infection would be an important addition to the armamentarium of the physician or veterinarian. Such a substance could be used prophylactically with subjects constantly exposed to infection, defensively with subjects about to undergo surgery, or supportively to improve the immunological response of subjects already suffering from an active infection or recovering from surgery or other trauma.
It has now been discovered that an immunostimulatory factor utilizable to achieve these results can be isolated from bovine thymus tissue.
This factor has a number of distinguishing characteristics by which it can be identified and distinguished from other stimulatory factors which have been described, including some which have been found in thymus tissue. Among these characteristics are the following:
1. It is amphoteric and strongly adheres to Sephadex and BioGel-P at both neutral and slightly acid pH.
2. Its isoelectric point is from 7.0 to 7.8.
3. It stimulates the DNA-synthetic response of both murine and human lymphocytes to alloantigenic stimulation in mixed lymphocyte culture (MLC).
4. It does not significantly increase the stimulation of splenic murine lymphocytes by lipopolysaccharide from Salmonella typhosa (LPS).
5. There is no decrease in its immunostimulatory activity in the presence of 2-mercaptoethanol.
6. Its stimulatory activity is fully reversible.
7. It functions more effectively in MLC medium containing human serum than in such medium containing fetal serum.
8. Its stimulatory activity in MLC is not affected by the presence of mouse serum.
9. It will accelerate the rejection of histoincompatible skin grafts in mice.
10. On dialysis through dialysis tubing with a 3500 Daltons cut-off, it passes through the tubing.
11. On gel filtration at pH 10.2 in 0.05M ammonium hydroxide, it splits into two immunostimulatory fractions, one with a molecular weight of about 1400 Daltons and the other less than 1400 Daltons but more than 100 Daltons.
The immunostimulatory factor of this invention is useful to stimulate the resistance of the mammalian or avian body to infection either as a preventative measure or as a curvature measure.
FIG. 1 is a Sephadex G-25 column profile of a calf thymus extract acetone precipitate. Fractions were screened for absorbance at 280 nm ( - - - ) and for activity in a murine MLC (--). The marker A indicates the elution position of bovine serum albumin; B, bacitracin; and C, sodium chloride.
FIG. 2 is a Sephadex G-50 column profile of a G-25 stimulator pool. The Sephadex G-50 column was equilibrated with phosphate buffered saline (PBS). A 5.0 mg sample of the G-25 stimulator pool was applied in 1 ml of PBS. The markers are A, bovine serum albumin; B, cytochrome C; and C, bacitracin. Fractions were screened for absorbance at 280 nm ( - - - ) and for activity in MLC (--).
FIG. 3 is a BioGel P-10 column profile of a G-25 stimulator pool. The BioGel P-10 column was equilibrated with 0.5M ammonium bicarbonate, pH 8.0. A 2.7 mg sample of G-25 stimulator pool was applied in 1 ml of elution buffer. The markers are A, bovine serum albumin and B, bacitracin. Fractions were screened for absorbance at 280 nm ( - - - ) and for activity in MLC (--).
FIG. 4 shows the effect of the passage of a calf thymus extract-acetone precipitate fraction through anion (Ag-1) or cation (AG-50) exchange columns on the ability to inhibit the MLC. AG-1 ( ) shows the effect observed with the fraction not retained by the AG-1 column; AG-50 ( ) shows the inhibition observed with the fraction not retained by the AG-50 column. Control ( - - - ) shows the inhibition observed with the untreated sample.
FIG. 5 illustrates ion exchange chromatography of the G-25 stimulator pool. A sample of G-25 stimulator pool, 4.5 mg, was applied to and eluted from a column of BioGel AG-50 ion exchange resin. The fractions were screened for absorbance at 280 nm ( - - - ) and for activity in the MCL (--).
FIG. 6 shows the effect of 2-mercaptoethanol concentration on the activity of the G-25 stimulator pool. A murine MLC was set up at various concentrations of 2-mercaptoethanol and a constant amount of G-25 stimulator pool, 20 μg/ml. The incorporation of 3 H-thymidine into control cultures ( ) and G-25 stimulator containing cultures ( ) was determined.
FIG. 7 shows the column profile of the G-25 stimulator pool on BioGel P-2 column. A 10 mg sample of the active pool was applied to a 1�45 cm column of BioGel P-2 equilibrated in 0.05M NH4 OH, pH 10.2 and eluted with the same reagent. Fractions were screened for absorbance at 280 nm ( - - - ) and for activity in the MLC assay (--). The markers are A, bovine serum albumin and B, sodium chloride.
The immunostimulatory factor of this invention has been isolated from bovine thymus tissue, specifically frozen calf thymus or dried, defatted calf thymus. The latter material is available from Viobin Corporation, Monticello, Ill.
In the first step of the isolation procedure, thymus tissue from either source in cut into small pieces, e.g. 1 cm2 and homogenized into 3 to 4 volumes of ice cold buffer at about 0� to 5� C. at pH 7 to 9, for example, 50 mM ammonium carbonate, pH 8.5 in a Polytron homogenizer for two minutes at low speed and two minutes at high speed. The homogenate is centrifuged at 26,000 to 30,000�G for 20 to 40 minutes at about 0� C. to 5� C., preferably 28,000�G for 30 minutes, and the resulting supernatant solution removed. The pellet is re-extracted as above and the pooled supernatants lyophilized. The lyophilized powder is suspended in water at about 0� C. to 5� C. at a concentration of 0.2 to 0.3 gm/ml and absolute ethanol is slowly added at the same temperature while stirring to a final concentration of 50% to 60% (V/V). The solution is stirred for 16 to 18 hours at 0� to 5� C. followed by centrifugation at the same rates and times mentioned above. The resulting clear supernatant is slowly added to 5 to 6 volumes of acetone precooled to about - 5� to -10� C. and maintained at -5� C. to -15� C. for 3 to 6 days. The precipitate is isolated, e.g. by centrifugation or filtration, resuspended in 100 to 200 ml of distilled water or in the same amount of 20 mM acetic acid and lyophilized at 0� to 5� C.
The material isolated by this procedure is normally a light tan sticky powder which contains the stimulatory factor of this invention as well as a number of inhibitory factors and other materials. The desired factor can be isolated from this fraction by gel filtration or by ion exchange chromatography. For convenience, it is referred to herein as the CTE-acetone precipitate.
The various procedures utilized in isolating, characterizing and establishing the utility for the stimulatory factor of this invention will now be discussed.
In one gel filtration procedure CTE-acetone precipitate is passed over Sephadex G-25 in a 90�2.2 cm column equilibrated with an acidic lyophilizable buffer at a pH of from 4 to 5 such as 0.1M acetic acid, 100 mM pyridine acetate, pH 4.5 or 0.1M ammonium acetate, pH 5.0. Two milliliter fractions are collected and assayed by MLC. The results with the pyridine acetate are shown in FIG. 1.
Other gel filtration procedures are carried out on a 1�45 cm column of Sephadex G-50 in phosphate buffered saline, (PBS) 0.02M phosphate, 0.15 sodium chloride, pH 7.4 and a 1�45 cm column of BioGel P-10 in 0.5M ammonium bicarbonate, pH 8.0.
For molecular weight determinations, the pooled stimulatory fractions from a G-25 column are passed over a selected BioGel column of the same dimensions at pH 10.2 in 0.05M NH4 OH and eluted with the same reagents.
The Sephadex products are a well known series of cross linked dextrans available from Pharmacia Fine Chemicals, Inc. of Piscataway, N.J. They are widely employed for the separation of natural materials. As normally utilized, the selected Sephadex is packed in a column, a solution containing the dissolved material to be analyzed is passed over the column and eluted with a selected solvent or series of solvents. Each specific Sephadex will separate materials of a specific molecular weight range. Dissolved materials above that molecular weight range will pass through the column in solution in the solvent front - - - the so called void volume. Materials which are retained will be successively eluted as more and more solvent is passed through the column. They will elute in order of progressively decreasing molecular weights.
Sephadex G10 will separate materials with molecular weights of from 200 to 700 Daltons.
Sephadex G-25 will separate materials with molecular weights of from 5000 to 1000 Daltons.
Sephadex G-50 will separate materials with molecular weights of from 30,000 to 1,500 Daltons.
BioGel P is a series of porous polyacrylamide beads used for high resolution gel filtration in a manner similar to Sephadex Gels. Available from Bio-Rad Laboratories, Richmond, Calif.
The ranges for the various species are as follows:
BioGel P-10 will separate 20,000 to 1,500 Daltons.
BioGel P-4 will separate 4,000 to 8,000 Daltons.
BioGel P-2 will separate 1,800 to 100 Daltons.
All gel filtration columns are calibrated with standards of known molecular weight and with sodium chloride. The standards employed in the procedures described herein are:
______________________________________bovine serum albumin 68,000 Daltonscytochrome C 12,000 Daltonsbacitracin 1,400 Daltons______________________________________
The standards are detected either by absorbance at 280 nm or by reaction with ninhydrin. The elution position of sodium chloride is determined by precipitation with acidic silver nitrate. The sodium chloride gives the position of the smallest molecules eluted from the column. In normal usage, all fractions below the lowest fractionation range will elute together with the sodium chloride.
Small scale ion exchange columns are prepared using Bio-Rad, AG-50 WX8 and AG-1-X8 resins, ammonia and acetate forms respectively. The resins are washed extensively and equilibrated with 10 mM ammonium acetate, pH 7.0. Small columns of approximately 8 ml volume are prepared in 10 ml plastic syringes fitted with Luer-lok valves. For analysis of the ionic nature of the stimulatory factor, 150 mg of the acetone precipitated product described above is dissolved in distilled water such that the conductivity is less than the 10 mM ammonium acetate, and the pH adjusted to 7 to 7.5. One third of this solution is applied to each column, while the remainder serves as a control. The sample is run slowly through the column, and the column is washed with 5 column volumes of 10 mM ammonium acetate, pH 7.0. The unretained fraction is lyophilized, redissolved in distilled water, again lyophilized, and subjected to MLC assay.
Bio-Rad AG-resins are crosslinked polystyrene beads in which some of the phenyl moieties are substituted with ionic functional groups. AG-50 is a strong cation exchange resin with sulfonic groups. AG-1 is a strong anion exchange resin with quaternary ammonium substituents. They are available from Bio-Rad laboratories.
Large scale ion exchange chromatography is performed on a 1�30 cm column of AG 50 WX8 resin, ammonia form, equilibrated with distilled water. Pooled samples of stimulatory composition from a G-25 column are dissolved in distilled water and adjusted to low conductivity and a pH of 5 to 6 with 0.01 acetate buffer. The column is eluted with a gradient made by mixing 90 ml each of distilled water and 1M ammonium hydroxide. Fractions of approximately 2 ml are collected, lyophilized, and assayed for activity by MLC.
Dialysis is performed using Spectrapor-3 dialysis tubing (Spectrum Medical Industries, Inc. 60916 Terminal Annex, Los Angeles 90054) with a manufacturer's determined molecular weight cut-off of 3,500 Daltons. At equilibrium, molecules with a molecular weight greater than 3,500 Daltons are retained within the dialysis bag (the Retentate) and those of molecular weight less than 3,500 Daltons will pass through the bag (the Dialysate). The relative amounts of activity at equilibrium will be in proportion to the relative volumes of each fraction, in these experiments 30 ml of Retentate to 300 ml of Dialysate. For a less than 3,500 molecular weight component, approximately 10% would be expected to be found in the Retentate and 90% in the Dialysate.
The spleen is sterilly removed from C57/BL and Balb/C mice and placed in a Petri dish containing cell collection media (Earl's Balanced Salt solution containing 2.5% heatinactivated fetal calf or human serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 100 IU/ml heparin). The spleen is perfused briefly using a 22- or 25-gauge needle to remove red blood cells, placed in a second dish, and teased gently to remove the spleen lymphocytes. The large pieces are removed through a 50-mesh screen and the lymphocytes are recovered by centrifugation at 200 G for 10 minutes. The cells are washed once with cell collecting media and resuspended incomplete assay media (Minimum Essential Media, Gibco), 5% or 10% heat inactivated human or fetal calf serum, and 5�10-4 M 2-mercaptoethanol. Human peripheral blood lymphocytes are obtained from blood donated by healthy laboratory personnel. Blood is defibrinated by shaking with glass beads and lymphocytes obtained by centrifugation through a Ficole-Hypaque cushion. The lymphocytes collect at the interphase, are washed by centrifugation with Earl's Balanced Salt Solution (Gibco) and suspended in complete assay media without added 2-mercaptoethanol.
Two-way MLC are set up by adding 1.25�105 cells/well from each strain or donor to microwell plates (Falcon No. 3040 or Linbro No. IS-FB-96). The plates are incubated at 37� C. for four days. Tritiated thymidine (0.5 μCi, 2Ci/nmole) is added for the last six hours of the culture period. At the end of the culture period, the extent of blast cell formation is obtained by microscopic examination of the plates. The cells are harvested using a Satron cell harvester, and the amount of radioactive label incorporated determined. The incorporation of 3 H-thymidine in control murine MLC in 10% human serum is 30,000 cpm per well with an average standard deviation in all samples of 10% or less of the total incorporation determined. The incorporation of 3 H-thymidine is a measure of blast cell formation and therefore a measure of immunological response.
For stimulation of various lymphocyte sub-populations by lectins, spleen cell suspensions are prepared as above and 250,000 cells of one strain are placed in each well. Experimentally determined optimum amounts of mitogens are added and the incubations continued for 72 hours. The lectins used are Con A (Difco, 0.1 μg/well), Phytohemaglutinin-M (PHA-M, Difco, 20 μg/well) and lipopolysaccharide from Salmonella typhosa (LPS, Sigma, 1.5 μg/well). The extent of blast cell formation and label incorporation are measured by the same procedures as described for MLC.
For this in vivo study of immune response, C57/BL recipients were grafted with full thickness grafts obtained from histoincompatible Balb/C mice. Grafts of about 1 cm2 are obtained from Balb/C donors and sutured at the corners to prepared beds on the backs of groups of six C57/Bl recipients under Halothane anesthesia. The grafts are inspected daily both visually and tactily starting on the sixth day after grafting. One set of animals is preimmunized by grafting and rejecting a similar transplant one month prior to the final experiment. Statistical analysis is in accordance with students' t-test for paired populations.
The results of the studies described above will now be described in detail so that those skilled in the art will recognize the characterizing features of the immunostimulatory factor of thsi invention.
The biological activities present in the CTE-acetone precipitate were resolved by gel filtration on columns of Sephadex G-25. The entire effluent of the column was screened by determining the effect of the various fractions on murine MLC in human serum. The use of human serum to culture the lymphoid cells was observed to be essential to avoid the strong inhibitory effects of polyamines present in the tissue extracts.
The results of the screening with pyridine acetate buffer at pH 4.5 are shown in FIG. 1 from which it will be seen that the inhibitory factor is eluted just after the bacitracin marker at a KAVE of 0.76.
The stimulatory factor elutes immediately behind the salt at a KAVE of 1.33 near a large 280 nm absorbing peak. The yield of stimulatory material obtained by this procedure was about 1.5 mg for each 1000 mg of CTE-acetone precipitate applied to the column.
Similar results were obtained with commercial defatted calf thymus.
From the elution position of the stimulatory factor it is clear that it must interact or adsorb strongly to Sephadex G-25 under the elution conditions as described above.
As shown in FIG. 2, the stimulatory factor is also strongly bound to Sephadex G-50 equilibrated with PBS at pH 7.4. It was observed in this study that the stimulatory factor eluted well beyond the molecular weight appropriate to other thymus factors which have been reported and to interleukin-2. The factor is also distinguished from these factors since they have not been reported to absorb to Sephadex under these conditions. These reports appear in:
Goldstein, G.: Nature 247: 11-14 (1974).
Kook, A. I., Yakir, Y., and Trainin, H.: Cell Immunol. 19: 151-157 (1975).
Dardenne, M., Pleau, J. M., Man, N. K., and Bach, J. F.: J. Biol. Chem 252: 8040-8044 (1977).
Low, T.L.K., Thurman, G. B., Chincanini, C., McClure, J. E., Marshall, G. D., Hu, S. K., and Goldstein, A. L.: Ann. N.Y. Acad. Sci. 332: 33-48 (1979).
Mier, J. W. and Gallow, R. C.: Proc. Natl. Acad. Sci. U.S.A. 77: 6134-6138 (1980).
Gillis, J., Snith, K. A., and Watson, J: J. Immunol. 124: 1954-1962 (1980).
Disabato, G.: Proc. Natl. Acad. Sci. U.S.A. 79: 3020-3023 (1982).
Watson, J., Gillis, S., Marbrook, J., Mochizuki, D., and Smith, K. A.: J. Exp. Med. 150: 849-858.
Thus, at pH 4.5 to 7.4, this stimulatory factor displays a property of chromatographic behavior on Sephadex gels which is distinctly different from previously described thymic or lymphokine factors.
A number of peptides possess the property of chemabsorbance on Sephadex gels. One of the techniques for minimizing this is to use a polyacrylamide gel matrix such as BioGel at an alkaline pH as described by Chin and Wold: Anal. Biochem. 16: 585 (1972). A sample of G-25 absorbed material was subjected to chromatography on BioGel P-10 run in 0.5M ammonium bicarbonate, pH 8.0. The elution pattern is shown in FIG. 3. The 280 nm absorbing material was held back under these conditions, similar to the results with the Sephadex G-25 and Sephadex G-50 columns. The stimulatory activity was also held back, indicating the strongly absorbing properties of the stimulatory factor.
From the above, it can be concluded that the stimulatory factor of this invention strongly adheres to any Sephadex at pH 4.5 to 8.0 and to any BioGel at pH 8.0.
When the technique of Chin and Wold was employed under strongly basic conditions, the stimulatory factor eluted before the salt on a BioGel P-2 column as shown in FIG. 7. In this experiment, 10 mg of G-25 stimulatory pool was passed over a 1�45 cm column of BioGel P-2 which had been equilibrated with 0.05M NH4 OH, pH 10.2. The fractions were eluted with the same reagent. One result of this treatment is that the factor is split into two fractions. The pattern of FIG. 7 suggests that one of these fractions has a molecular weight of about 1400 Daltons and the other a molecular weight of less than 1400 Daltons but more than 100 Daltons. The significance of the appearance of the two fractions under these strongly basic conditions is not completely understood. It may be that the factor of the invention actually contains two fractions, or that it is split into two fractions by the strong alkaline treatment.
The term `strongly adheres` as used in this specification and claims means that the factor elutes after a sodium chloride marker on a Sephadex column at pH 4 to 8.
For the dialysis study, a sample of G-25 pool was taken up in 0.1M ammonium acetate, pH 5.0. The Retentate and the Dialysate were freeze dried and assayed by MLC. It was found that 13% of the activity was in the Retentate and 87% of the activity was in the Dialysate. This clearly establishes that the immunostimulatory factor passes through dialysis tubing with a 3500 Dalton cut-off.
The charge nature of the stimulatory activity has been investigated in several experiments. In the first, the CTE-acetone precipitated material was subjected to analysis on small columns of ion exchange resins. The CTE-acetone precipitated fraction was applied to small columns of either an anion (AG-1) or a cation (AG-50) exchange resin at pH 7. The fraction not retained by the column was examined for its effects on the MLC. As shown in FIG. 4, the fraction not retained by the AG-1 resin significantly stimulated the 3 H-thymidine incorporation in the MLC. Microscopic examination revealed obvious increases in the number of blast cells at the end of the culture period compared to the control sample. The fraction not retained by the AG-50 column showed an almost threefold increase in total inhibitory activity. This was apparently the result of the removal of stimulatory activity present in the crude CTE-acetone precipitate fraction. This also confirms the results with the G-25 columns which indicated the presence of both stimulatory and inhibitory activity in the extracts.
The G-25 stimulatory pool was also subjected to treatment on the ion exchange resin, AG-50, equilibrated with distilled water. The elution profile is shown in FIG. 5. The stimulatory activity was clearly separated from the bulk of the 280 nm absorbing material which was not retarded by the column under these conditions. These results indicate that the stimulatory factor does not bind to an anion exchange resin and is held slightly back by a cation exchange resin. From those results it can be concluded that the stimulatory factor has a neutral or slightly basic isoelectric point, i.e. about 7.0 to 7.8.
The G-25 stimulatory pool increases the DNA-synthetic response of both murine and human lymphocytes to alloantigens in the two-way MLC. The stimulation can be significant, up to five-fold, depending on the culture conditions. The average 2-fold stimulatory dose at the G-25 stage is 10 to 20 μg/ml for a murine MLC in 10% human serum. As shown in Table 1, the response of murine and human lymphocytes in MLC and of murine lymphocytes to a suboptimal dose of ConA was significantly stimulated. The stimulation of murine lymphocytes by LPS was not significantly increased by the presence of the stimulator. Clearly, the stimulator only works on T-cell mediated responses.
The G-25 stimulatory pool also has an effect on the proliferation of the murine leukemia, L1210, as shown also in Table 1. This indicates that the factor can have a direct mitogenic effect on continuously dividing cells.
TABLE 1______________________________________Effect of the G-25 stimulator pool on the lymphocyte proliferativeresponse to antigens and mitogens and on murine leukemic cells.1 3 H--CPM/well S.D. +G-25Cells Source Control Stimulator % Diff.______________________________________Lympho-cytesMLC murine 36,000 � 4,700 73,400 � 10,700 +99MLC human 20,850 � 2,100 45,600 � 5,100 +118Con A murine 9,760 � 1,450 8,790 � 1,170 -10LeukemicCellsL1210 murine 40,300 � 2,300 85,900 � 6,000 +113______________________________________ 1 Measured by increased counts attributable to tritiated thymidine i blast cells. Each assay was performed with a concentration of G25 stimulator pool known to cause a 2fold increase in the 3 H--thymidin incorporation of a murine, two way MLC in 10% human serum.
It has been shown that murine lymphocytes require thiol compounds for maximal response, (Broome et al.: J. Exp. Med. 138: 574 (1973). One way a stimulatory factor could function could be to modify in some way these necessary thiols. To investigate this possibility a murine MLC was set up with various concentrations of 2-mercaptoethanol and contrasted with a concentration of G-25 stimulator pool previously shown to produce a 2-fold stimulation under standard assay conditions. These results are shown in FIG. 6 which indicates that at any 2-mercaptoethanol concentration at which there is a 3 H-thymidine incorporation significantly above background, that is, a significant response to the alloantigens by the spleen cells, the stimulator increased the response. The stimulator was also active over a wide range of 2-mercaptoethanol concentrations. This shows that alteration of thiol levels is not involved in the action of the stimulator. It also confirms the results with the human MLC which does not require 2-mercaptoethanol in the media for reactivity.
Experiments in which various amounts of the G-25 stimulator pool were added at various times or removed at various times subsequent to the initiation of the MLC are shown in Table 2, 3 and 4. The results show that the action of the stimulator was reversible up until the point that microscopically visible lymphoblast cells are present in the cultures, 72 hr (Tables 2 and 3) and that the stimulator need only be present during the last 24 hr. or less of the culture period (Table 4). All these results are consistent with the target cell of the stimulatory factor being the lymphoblast itself. This clearly distinguishes this factor from other thymic factors which mainly stimulate the maturation of immature T-cells.
It has been observed that the G-25 stimulator pool seemed to work less well in two-way MLCs in medium containing 10% fetal calf serum than in medium containing 10% human serum. When a two-way MLC of murine lymphocytes was conducted in 5% fetal calf serum, under which conditions the total thymidine uptake is about 60% of that which can be demonstrated at 10% fetal calf serum, the stimulator could now increase the total thymidine uptake of these cultures to that observed at 10% serum. In 1% FCS, the G-25 stimulator pool at a concentration of 40 μg/ml could increase the response to that observed in 20% FCS. In this manner, the stimulator can partially replace the serum requirement for the MLC.
It has been shown that mouse serum will inhibit the efficacy of T-cell growth factor, Interleukin-2, Hardt et al. Various concentrations of pooled mouse serum along with various concentrations of the G-25 stimulator pool have been added to two-way murine mixed lymphocyte culture in 5% human serum. There is no evidence that mouse serum at any concentration had an inhibitory effect against the stimulatory ability of any concentration of the stimulatory factor. This serves further to distinguish between the thymic stimulator of this invention and Interleukin-2.
An important aspect of the immune stimulatory factor is its activity in vivo. The ability of the G-25 stimulator pool to accelerate the rejection of histoincompatible skin grafts in mice has been examined. The results are shown in Table 5. At two different doses, the partially purified stimulator significantly accelerated the rejection of the grafts. In the second experiment, the treated mice rejected the grafts at a rate comparable with preimmunized controls. At the doses used, there were no apparent toxic effects.
TABLE 2______________________________________REVERSIBILITY OF G-25 STIMULATOR IN MLC AT 24 HR. 3 H--CPM/Well S.D. (n = 6)Concentration Washed atμg/ml Control + 24 hr.______________________________________ 0 31,200 � 1600 35,300 � 270060 64,700 � 7200 32,700 � 220030 57,100 � 4800 28,500 � 250015 51,900 � 5900 36,300 � 3200______________________________________
The reversibility was determined by incubating 1�106 spleen cells of each type in 1.6 ml standard culture media plus or minus the indicated concentration of G-25 stimulator pool in 3 ml plastic culture tubes. After 24 hr., all the tubes were centrifuged at 200�g for 10 min. to pellet the cells and the media was aspirated from one set of tubes and replaced with an equal volume of prewarmed media without stimulator. These tubes were centrifuged and the media replaced again. After this procedure, the tubes were returned to the incubator for an additional 72 hr. At the end of the incubation, the cells were resuspended and 0.2 ml aliquots placed in microtiter wells for microscopic examination, labeling, and harvesting by the usual procedure.
TABLE 3______________________________________REVERSIBILITY OF G-25 STIMULATOR IN MLC 3 H--CPM/Well S.D. (n = 6) G-25 Stimulator Conc.Treatment 78 μg/ml 34 μg/ml______________________________________Control, no 40,100 � 6300 40,100 � 6300stimulatorControl, + 67,600 � 3200 86,100 � 2500stimulatorWashed, + 48 hr 29,800 � 1600 35,900 � 600Washed, 72 hr 44,900 � 1500 46,000 � 500Washed, + 96 hr 58,700 � 700 69,200 � 1900______________________________________
The reversibility was determined as described under Table 2 except the cells were washed at the indicated times.
TABLE 4__________________________________________________________________________Time course of addition of G-25 Stimulator to MLC 3 H--CPM/Well S.D. (n = 6)Time of G-25 StimulatorAdditionControl (PBS) 70 μg/ml 35 μg/ml 18 μg/ml__________________________________________________________________________0 73,000 � 7,200 167,600 � 15,600 159,500 � 7,500 145,500 � 800+ 24 hr75,400 � 10,100 161,100 � 8,500 150,900 � 10,100 145,700 � 13,200+ 48 hr66,300 � 7,300 173,700 � 10,000 148,100 � 11,700 119,700 � 7,900+ 72 hr67,700 � 9,600 166,500 � 12,900 139,400 � 21,000 105,500 � 6,500__________________________________________________________________________ Standard murine MLC assays were set up and the indicated amount of G25 stimulator pool was added at the indicated time after initiation of the culture. The cells were labeled after the last addition.
TABLE 5______________________________________In Vivo Activity of G-25 Stimulator: Accelerationof BALB/c skin graft rejection by C57/BL mice Day RejectedTreatment Ave. S.D. Diff. Sig.______________________________________A. 40 μg/I.P./Daily Treated 9.5 .4 0.8 .05 Control 10.3 .7B. 360 μg/I.P./Daily Treated 8.8 1.2 2.7 .01 Control 11.5 .6 2.3 .005 Pre-immunized control 9.2 .8______________________________________ Skin grafts were performed as described herein. Treated mice received the indicated amounts of the G25 stimulator pool. Significance was determined using the Students Ttest.
The isolation and biological characterization of a new immunostimulatory factor from bovine thymus has been described. In the presently preferred isolation procedure this factor is extracted from fresh or commercially defatted and dried thymus preparations with a basic buffer followed by solution in 60% ethanol and precipitation by cold acetone. Purification can be effected on Sephadex or BioGel columns at selected hydrogen ion concentrations. The biological activity of the factor, even when only partially purified, resides in its ability to stimulate both the DNA-synthetic response and the number of resulting blast cells of both murine and human T-lymphocytes responding to alloantigen stimulation.
The G-25 stimulator pool had no apparent effect on murine B-lymphocyte response to the polyclonal mitogen LPS. The reversibility experiments described above show that the factor acts very late in the MLC, after or during the formation of morphologically recognizable lymphoblasts in the cultures. The stimulatory effects are totally reversible if the factor is removed before the expiration of 72 hr. of the MLC, but not after 96 hr. In addition, the factor will stimulate the MLC if it is added as late as 72 hr. after start up. The stimulation achieved is equal to the level of stimulation obtained if the factor is continuously present.
The most important biological activity of the factor is the ability to stimulate an in vivo immune response. In the model system employed, the treated mice rejected allogenic grafts at a rate comparable to that of pre-immunized animals. This is probably the maximal rate obtainable in this type of strongly histoincompatible system. It should be noted that these results were obtained in fully immunocompetent, adult mice, responding to a strong antigenic stimulus.
The chemical and biological results described herein show that the stimulatory factor of this invention is distinct from other known thymic hormones and from Interleukin-2. The following Table 6 summarizes the most significant of these distinctions.
TABLE 6__________________________________________________________________________Comparison of some properties of various thymic factors and Interleukin21 FACTORPROPERTY G-25 Stim. FTS2 THF3 Thymopoietin Thymosin V IL-24__________________________________________________________________________MLC5 stimulates no effect inhibits/ stimulates inhibits stimulates stimulatesReversibi- yes no no no no yeslity6 at +24 hr.Gel Filtra- Adsorbs Free Free Free Free Freetion7Charge8 slightly neutral acidic acidic mixture acidic basic__________________________________________________________________________ 1 Data are from references mentioned below. 2 Facteur Thymique Serique Dardene et al: J. Biol. Chem. 252: 8040 (1977) 3 Thymic Humoral Factor Kook et al: Cell Immunol. 19: 151 (1975) 4 Interleukin 2 or Tcell Growth Factor Disabato: Proc. Natl. Acad. Sci. U.S.A. 79: 3020 (1982) 5 Refers to activity in a MLC with either murine, spleen or human peripheral lymphocytes. Several authentic samples of FTS were without effect in MLC system described herein. THF stimulates an MLC only if washed from the cells within 24 hr., if continuously present, THF inhibit 6 Reversibility determined by washing from cells within 24 hr. or after preincubation. 7 Refers to elution behaviour on various gel filtration supports and under at least one condition mentioned herein. 8 Refers to the ionic nature determined by isoelectric focusing, electrophoresis, or ion exchange chromatography.
The immunostimulatory factor of this invention can be obtained in highly purified form in accordance with the procedures described herein. However, it is not essential that there be complete purity to achieve the desired therapeutic effect. Compositions containing the factor can be isolated and used alone or in various pharmaceutical compositions to achieve a therapeutic level of the factor in mammalian or avian blood or tissues which is effective to generate an immunostimulatory response.
Several different commercially available factors with immunostimulatory activity were tested in the MLC system. The results of the studies are shown in Table 7. The dosages were selected so as to cover the range which appeared to be optimal based on study of the publications shown in the table and other standard considerations.
TABLE 7______________________________________Comparison of the effect of various factorson the standard MLCFactor Dosage % Increase______________________________________a saline -- --b G-25 5 μg/ml +301c IL-2 271/2 Max units +43d EGF 3 ng to 6.7 μg/ml 0e Thymopoietin II 30 ng to 67 μg/ml 0f FTS 7 ng to 16 μg/ml 0g FTS + Zn 7 ng to 16 μg/ml 0h Tufsin 70 ng to 167 μg/ml 0i Levamisole 1.3 � 10-9 M to 2.8 � 10-4 M 0______________________________________ a control MLC value with only saline addition b Standard G25 active immunostimulator pool c Interleukin-2, collaborative Research, obtained from Con A stimulated RatTable supernatant by ammonium sulfate precipitation and DEAESephadex. Units as defined by activity against mouse cytotoxic Tlymphocyte line [Rascett et all Blood 57, 399 (1981); Mier et al Proc. Natl. Acad. Sci. U.S.A. 77, 6134 (1980); Di Sabato Proc. Nat. Acad. Sci. U.S.A. 79, 3020 (1982)]- d Epidermal Growth Factor, Collaborative Research. No reported immunomodulatory activity but does adsorb to BioGel P10, Savage et al, J. Biol. Chem. 247, 7609 (1972) e Thymopoietin II active peptide fragment, United States Biochemicals, synthetic peptide [Goldstine Nature (London) 274, 11 (1974) Schlesinger et al, Cell 5, 367 f & g Facteur Thymique Serique, synthetic peptide, Penninsula Labs. The complex with Zn is the reported active form for the synthetic peptide [Dardenna et al, J. Biol. Chem. 252, 8040 (1977); Bach et al, Trans. Proc XIV 509 (1982)]- h Tufsin, Vega Biochemicals, synthetic tetrapeptide, shown to stimulate macrophages, Nishioka et al, Biochem. Biophys. Res. Comm. 47, 172 (1972) i Levamisole, Sigma Chemical Co., synthetic compound with immunomodulating properties, Hadden, J. W. in The Pharmacology of Immunoregulation. Eds. Werner, G. H. and Floc/h, F. Academic Press, (1978 page 369 et seq.
As will be seen from the table, only the lymphokine IL-2 showed any enhancing effect. In fact, as will be seen from the next table, Table 8, there is a very strong synergistic effect of the two factors.
TABLE 8__________________________________________________________________________Synergistic Effect of G-25 Immunostimulator and IL-2Factor Concentration 3 H--CPM/Well S.D. % Increase % Synergy__________________________________________________________________________(a) saline control -- 13,100 � 1,670 -- -- G-25 15 μg/ml 44,400 � 4,000 239 -- IL-2 30 U/ml 25,600 � 2,300 20 -- G-25 + IL-2 15 μg/ml + 30 U/ml 85,900 � 16,500 556 316(b) saline control -- 12,300 � 1,200 -- -- G-25 33 μg/ml 30,000 � 3,100 144 -- IL-2 15 U/ml 18,800 � 1,300 52 -- G-25 + IL-2 33 μg/ml + 15 U/ml 70,600 � 5,600 474 330__________________________________________________________________________ Standard MLG assays were setup with the indicated concentrations of each factor. The % increase = 100 � (Experimental cpmsaline control cpm)/saline cpm. The % synergy is defined as 100 � (cpm with G25 + IL2-cpm with G25 alone)/saline control cpm.
It is apparent that compositions containing both the immunostimulatory factor of this invention and IL-2 are valuable for the treatment of patients in need of immunostimulation. Such compositions will typically contain from 10% to 90% by weight of IL-2 based on the total weight of the immunostimulatory factors. The composition may contain any of the usual pharmaceutical excipients.
The products of this invention, that is the factor in highly purified form, salts of the factor or isolated fractions containing the factor, are useful mammalian and avian therapeutic agents and are effective for stimulating an immune response with patients in need of such treatment.
The immunostimulatory factor of this invention is useful for the treatment of any avian or mammalian disease associated with depression of cell mediated immunity. Severe viral, bacterial and fungal infections are often associated with such depression. The factor can be used to enhance the effects of standard therapy normally employed for such infections.
The factor is especially valuable to assist in stimulating the immune response of patients undergoing radiation or chemotherapy for the treatment of malignant diseases.
It is also useful for protecting subjects under such stress as to render them susceptible to infections which they are normally able to resist. Typical examples of such utility are protection of animals against shipping fever, or protection of humans subsequent to surgery.
The physician or veterinarian will determine the dosage which will be most suitable for a particular application. It may vary from patient to patient depending on the size of the patient, the condition under treatment and other factors which are readily evaluated by those skilled in the art. For continuous administration over extended periods to individuals with severe or minor metabolic abnormalities or spleenectomized individuals, the product will normally be provided in various dosage forms varying from relatively large to build up a prompt blood level to relatively small to maintain an effective level. For intermittent treatments to combat acute or chronic infections various dosage forms may be provided.
Normally, the dosage will be in the range of from 0.1 to 0.5 mg/Kg of body weight. To provide these levels, dosage unit forms containing from 0.05 to 0.25 mg of active product can be prepared. Dosage unit forms may, as indicated above, additionally contain from 0.005 to 0.225 mg of IL-2.
The products of this invention may be administered alone but will generally be administered with pharmaceutically acceptable, non-toxic carriers, the proportions of which are determined by the suitability and chemical nature of the particular carrier, the chosen route of administration, and standard pharmaceutical practice. For example, in combatting various infections or in maintaining therapeutically effective levels in the blood or tissue, intravenous or intramuscular administration may be used. The compositions may be in the form of sterile solutions containing other solutes, for example, enough saline or glucose to make them isotonic. Those skilled in the art will know of many standard procedures for preparing such compositions.
An advantage arising from the amphoteric nature of the factor of this invention is that it can be utilized in the form of pharmacologically acceptable salts which may be either metallic salts or acid addition salts. These salts have the advantage of water solubility and are particularly useful for parenteral administration. The metallic salts, especially the alkali metal salts, are relatively stable and for that reason are preferred over acid addition salts. The sodium salts are especially preferred because of their ease of preparation.
The acids which may be used to prepare the pharmacologically acceptable acid addition salts of this invention are those containing non-toxic anions and include, for example, hydrochloric, sulfuric, phosphoric, acetic, lactic, citric, tartaric, oxalic, succinic, maleic, gluconic and saccharic acids.
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|Gideon Goldstein
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|Bach et al., Transplant Proc., XIV:3, 509 512 (1982).
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|Broome et al., J. Exp. Med., 138, 574 592 (1973).
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|Dardenne et al., J. Biol. Chem., 252:22, 8040 8047 (1977).
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|Nishioka et al., Biochem. Biophys. Res. Comm., 47:1, 172 179 (1972).
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|Sep 23, 1988
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|Method of treating complications in immunodepressed states resulting from HIV infection
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|Apr 8, 2003
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|Nov 27, 2003
|Khan Nisar Ahmed
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|Apr 8, 2003
|Nov 27, 2003
|Khan Nisar Ahmed
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|Dec 4, 2003
|Khan Nisar Ahmed
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|Gert Wensvoort
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|Jul 15, 2004
|Erasmus Universiteit Rotterdam
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|Oct 14, 2004
|Khan Nisar Ahmed
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|Jan 7, 2004
|Oct 21, 2004
|Khan Nisar Ahmed
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|Oct 13, 2005
|Robbert Benner
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|Sep 27, 2005
|Feb 9, 2006
|Neorx Corporation, Regents Of The University Of Michigan
|Radiolabeled annexins
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|Robbert Benner
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|Mar 27, 2008
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|Mar 27, 2008
|Khan Nisar A
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|Oct 30, 2007
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|Oct 30, 2007
|Oct 2, 2008
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|Dec 25, 2008
|Khan Nisar A
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|Apr 9, 2009
|Sep 10, 2009
|Biotempt B.V.
|Methods and uses for protein breakdown products
|Oct 24, 2008
|Nov 12, 2009
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|Control of radiation injury
|Nov 26, 2009
|Biotempt B.V.
|Control of radiation injury
|Jan 13, 2011
|Biotempt B.V.
|Control of radiation injury
|Apr 14, 2009
|May 5, 2011
|Biotempt B.V.
|Gene regulator
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|Apr 10, 2012
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|Mar 9, 1993
|Sep 16, 1993
|Beardsley Terry R
|Immune-enhancing agent for therapeutic use in immunocompromised hosts
|U.S. Classification
|514/21.92, 530/837, 530/351, 424/85.2, 530/300
|International Classification
|Cooperative Classification
|European Classification
|Jul 23, 1986
Owner name: IMMUNOGENICS CORPORATION, 1000 SENECA STREET, SEAT
Free format text: ASSIGNS THE ENTIRE INTEREST, SUBJECT TO AGREEMENTS RECITED;ASSIGNOR:ENDORPHIN INC.;REEL/FRAME:004578/0255
Effective date: 19860616
|Sep 2, 1986
|Certificate of correction
|Sep 19, 1989
|Maintenance fee reminder mailed
|Feb 18, 1990
|Lapse for failure to pay maintenance fees
|Jun 19, 1990
|Expired due to failure to pay maintenance fee
Effective date: 19900218


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- Report: #774773
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Complaint Review: Jane Wilson
Jane Wilson840 Murdocksville Rd. West End, North Carolina United States of America
Jane Wilson EBAY ID: lowridin_momof2 Scammed me out of money from gift card via paypal reversal scheme, ignore positive feedback West End , North Carolina
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A couple of days later I received an email that stated the payment was being reversed by the bank meaning the buyer had 'insufficient funds' The case is now open in Paypal as of 9/9/11 The gift card is used and the buyer has been unresponsive. There is no way to freeze or stop the gift card. I have read much about the paypal reversal scam and I am trying to do everything in my power to make sure 'Jane' never cons another dime out of anyone. Do not trust and buyer beware. If you are ripped off, fight fight fight!
I am going to contact the local authorites or at least the NC ones if nothing can be done. This person will not scam another, EVER. I have screencapped our entire transaction and have not deleted anything. This person will pay, although the amount is 50.00 the buyer will not get one over on me. I urge all that have been scammed to report it to ebay, ic3.gov, paypal, whoever will listen. My local authorities will be hearing from me, as well as small claims court.
This report was posted on Ripoff Report on 09/09/2011 09:56 AM and is a permanent record located here: http://www.ripoffreport.com/r/Jane-Wilson/West-End-North-Carolina-27376/Jane-Wilson-EBAY-ID-lowridinmomof2-Scammed-me-out-of-money-from-gift-card-via-paypal-rev-774773. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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11 years of experience
Fort Stewart, GA 31314 Phone number & directions
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
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Dr. David Dobson, MD is board certified in Psychiatry.
- He has special expertise in 8 areas:
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Education & Awards
Dr. Dobson graduated from Loma Linda University.
Dr. Dobson has 1 hospital affiliations:
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- Dr. David Lee Dobson MD is a male Psychiatrist, has 11 years of experience and practices in Psychiatry.
- Where does Dr. David L. Dobson practice?
- Dr. David Dobson has 1 office location.
Dr. David Dobson practices Psychiatry near Fort Stewart, GA.
See Fort Stewart Psychiatrists.


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Price: £399.00Product ID: 733376
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This shower door folds inwards using its unique hinge mechanism and is suitable for recess fit or you could combine the door with a side panel to form a complete shower enclosure.
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Created in this Craftsy class
Pre-Cut Piecing Made Simple taught by Camille Roskelley
Camille Roskelley teaches her tricks for a quick start to quilting success. Even brand-new quilters will see immediate results using pre-cut fabrics.
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Very nice! I am ready to start my blocks now:)
07/06/2013 Flag
So perfect. I am using this as my inspiration block to built my swoon quilt colours around. My desire to use the happy go lucky navy floral was confirmed when I saw how beautiful your block turned out! Thank you for sharing. :-)
07/16/2013 Flag


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Hickey Freeman 'Beacon' Classic Fit Plaid Sport Coat
Was: $1,095.00
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Men's Ted Baker London 'Tom' Trim Fit Wool
Richly textured Italian wool elevates a smart blazer crafted in a versatile two-button cut framed with sharp notch lapels. Color(s): burgundy. Brand: TED BAKER LONDON. Style Name:Ted Baker London 'Tom' Trim Fit Wool Blazer. Style Number: 5156985. Available in stores. Learn More
Men's Good Man Brand Trim Fit French Terry
Remarkably soft French-terry cotton is tailored into a handsome Italian blazer cut slim with classic notch lapels and a two-button front. Mélange-woven yarn accents the neat, contemporary style. Color(s): grey heather. Brand: Good Man Brand. Style Name:Good Man Brand Trim Fit French Terry Blazer. Style Number: 5110253. Available in stores. ... Learn More
Men's W.r.k 'Keaton' Trim Fit Blazer, Size 40
Tailored for a lean frame in a soft fabric blend that features cotton and linen, this unstructured two-button blazer is fashioned for lightweight comfort and styled with a handsome notch lapel. Color(s): charcoal, light grey. Brand: WRK. Style Name:W.r.k 'Keaton' Trim Fit Blazer. Style Number: 5152330. Available in stores. Learn More
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Narrow notch lapels and low-profile welt pockets accentuate the clean, streamlined design of a distinguished two-button sport coat tailored from a substantial knit with subtle geometric texture. Color(s): navy. Brand: PS PAUL SMITH. Style Name:Ps Paul Smith Extra Trim Knit Cotton Blazer. Style Number: 5157975. Available in stores. Learn More
Men's Canali Classic Fit Solid Wool Blazer, Size
Premium Italian wool enhances a two-button blazer, detailed with a subtle textured weave. Tonal pickstitching marks the crisp notch lapels for a fine finishing touch. Color(s): black, navy. Brand: CANALI. Style Name:Canali Classic Fit Solid Wool Blazer. Style Number: 265931. Available in stores. Learn More
Men's Singer + Sargent Regular Fit Seersucker Cotton
Maintain an elegant profile in warmer weather with an impeccably tailored two-button jacket crafted from breathable cotton seersucker in a classic pairing of fresh blue and crisp white. Color(s): bone. Brand: Singer + Sergant. Style Name:Singer + Sargent Regular Fit Seersucker Cotton Blazer. Style Number: 5164511. Available in stores. Learn More
Men's Ted Baker London 'Bandman' Extra Trim Fit
Tailored in an extra-slim fit with a classic two-button closure, a microtexture blazer is detailed with branded buttons, a custom lapel pin and coordinating accents including a pop-up pocket square and a sharp contrast lining. Color(s): navy. Brand: TED BAKER LONDON. Style Name:Ted Baker London 'Bandman' Extra Trim Fit Blazer. ... Learn More


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This image provided by the Los Angeles County Superior Court shows Anna Nicole Smith with her daughter, Dannielynn and Howard K. Stern. Suggesting pictures are more powerful than words, the defense in the Anna Nicole Smith drug conspiracy case put on a slide show for jurors Friday of 90 photos depicting the last five months of the former Playboy model's life. With defense lawyers planning to call no witnesses after the prosecution rests its case Monday, the photos were their only evidence, designed to show Smith was not impaired by drugs as witnesses have testified.(AP Photo/ Los Angeles County Superior Court)


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- Report: #893085
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Complaint Review: Corporate Security Alliance
Corporate Security Alliance240 West 37th Street, NY, NY 10018 Internet United States of America
Corporate Security Alliance CSA Corporate Security Alliance Scam Internet, Internet
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This report was posted on Ripoff Report on 06/05/2012 06:45 AM and is a permanent record located here: http://www.ripoffreport.com/r/Corporate-Security-Alliance/internet/Corporate-Security-Alliance-CSA-Corporate-Security-Alliance-Scam-Internet-Internet-893085. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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I have continual trouble with air leaks on my aluminum wheels. The shop always tells me that it's due to corrosion between the wheel and tire bead.
Is there any way to recondition the wheel to stop this?
Thanks! Dave
Has the shop tried bead sealant? It gooey black stuff, you put it on the bead when installing the tire. Should normally seal stuff like that unless the leak is really bad.
-- Stephan 2001 Yamaha YZF-R6
Some 80's to early 90's Ford Aluminum wheels had a porosity problem. Ford has a TSB on it and a fix that used epoxy paint to seal the inside of the wheel.
I had a set of centerline aluminum wheels from the late 70's that had a leak between the 2 halves. I had the tire shop dismount the tire for me, took it home and removed the 25 year old silicone bead from the center of the wheel and applied new silicone. I brought it back to the tire shop and had them remount the tire and it has worked without a leak ever since, 2 years.
Dave Wick wrote:
Many tire shops will use a bead sealer on aluminums rims... it essentially glues the tire to the rim. Dismount the rubber, tidy up the rim with sandpaper or a wire brush to remove any loose corrosion and have them re-mounted using bead sealer. you can do it yourself if you like, just chalk line the tire to the rim so you don't lose the balance. You can do it without dismounting the tires but it's a pain in the ass to clean the rim while trying to hold the bead down - not to mention the crap will stay inside your tire.
Spray on bead sealer (never tried, can't vouch for it) http://www.rockcrawler.com/features/newsshorts/02september/safetyseal.asp
Brush on bead sealer (this is what the shops use) http://www.annco.com/products/pro_2.cfm?ItemNumber=G10106
Take off tire and clean up bead area either with a fine wire wheel on a drill, or fine sandpaper, to polish up that area while taking off the corrosion. I have heard of different sealers like Form A Gasket #2 (IIRC an old racer's trick), bead sealer, etc. I have heard of people painting the bead area to stop further corrosion using clear or POR-15 or ??? This makes sense to me. ;-)
I think I only did the fine wire wheel to clean up the Canyon alum wheels now on my 88 XJ about 3-4 years ago. I did not paint nor use a sealer, but I think I applied a little ATF to get the Duellers on the wheels though.
later, dave AKA vwdoc1
They can brush it with a wire brush in a drill bit, kind of like a Dremel tool.
Do Not use those Seal-A-Leak cans of goo. I had a bead leak several years ago and used some of that shit and it rusted the inside of my rim.
You might want to post this in a welding or mechanical engineering newsgroup or go to a good machine shop. I'm thinking - clean it up real good and spray weld it, but I'm talking out of my field.
not wheels expert here but since when does aluminum rust? - or is there something else there besides aluminum?
Just the terminology.. iron oxidizes = rust, aluminum oxidizes = dunno whut its called. As with all metals, some grades are more porous than others and when they oxidize or corrode the pores get bigger quicker. Salt promotes corrosion, which is the main reason us Northerners remove the aluminum rims in the winter.... an lemme tell ya.. its a real bitch getting traction with just the rotors.
Jafo proclaimed:
Corundum. The main ingredient in sapphire and other rather hard and clear gemstones. Or watch crystals.
Rusting/corrosion is an oxidizing process. Hence aluminum can oxidize and the "rusting" is aluminum oxide. Iron/steel rusting would produce ferrous oxide. The colors are different - iron -red/brown and aluminum white/gray.
Richard proclaimed:
Aluminum oxide in the absence of other substances is extremely clear. Most aluminum corrosion is due to acetates, oxylates, chlorides/ates, etc.
Aluminum rust as such is sapphire. However aluminum corrodes quite nicely in a variety of ways.
For the OP, have the rims polished and you might think about also having the inside [where the air goes] heavily painted. Been a long time since I ran into stories of such, but some of the early cast style aluminum wheels were rather porous. Thus the famous slogan "Stop Casting Porosity"
Peter D. proclaimed:
Motorsforum.com is a website by car enthusiasts for car enthusiasts. It is not affiliated with any of the car or spare part manufacturers or car dealers discussed here. All logos and trade names are the property of their respective owners.


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- Report: #907062
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Complaint Review: Homeward Residential
Homeward ResidentialInternet United States of America
Homeward Residential Homeward Residential is the Worst i've ever delt with. What a Nightmare. Internet
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This report was posted on Ripoff Report on 07/05/2012 09:50 AM and is a permanent record located here: http://www.ripoffreport.com/r/Homeward-Residential/internet/Homeward-Residential-Homeward-Residential-is-the-Worst-ive-ever-delt-with-What-a-Nightm-907062. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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