Novel galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases

a technology of galectin and composition, applied in the field of new galectin sequences, can solve the problems of complex discrimination between “harmful” inflammatory cells, inability to identify and treat “harmful” inflammatory cells, and inability to achieve the effect of reducing the inflammatory response in the individual

Inactive Publication Date: 2006-01-12
YISSUM RES DEV CO OF THE HEBREWUNIVERSITY OF JERUSALEM LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] According to yet an additional aspect of the present invention there is provided a method of reducing inflammatory response in an individual, com...

Problems solved by technology

Deformation of joints and disability are very common, but the disease may also decrease expected life span by an average of ˜9 years in the more active forms.
Selective therapeutic targeting of pathological cells, such as inflammatory cells is a major challenge for modern medicine.
Unfortunately, the existence of such discrete target entities in pathological cells is not an obvious phenomenon.
Discrimination between “harmful” inflammatory cells, involved in self-destruction, and inflammatory cells engaged in the eradication...

Method used

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  • Novel galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases
  • Novel galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases
  • Novel galectin sequences and compositions and methods utilizing same for treating or diagnosing arthritis and other chronic inflammatory diseases

Examples

Experimental program
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Effect test

example 1

Cloning of Galectin-8 and Galectin-8 Variants

[0269] Materials and Methods

[0270] A. Cloning of Galectin-8

[0271] RNA Preparation: Total RNA of mononuclear cells (MNC) derived from synovial fluids of RA patients was extracted using the Qiagen RNeasy kit (Qiagen, USA).

[0272] RT-PCR: 1 μg of the extracted RNA was reverse transcribed, and PCR-amplified using M-MuLV reverse transcriptase (first strand cDNA synthesis kit, Pharmacia) and Taq DNA polymerase (2.5units) (Gibco-BRL). The reactions were carried out in a DNA thermal cycler 480 (Perkin Elmer Cetus) in a final volume of 50 μl using oligonucleotide primers complementary to a translated region of human galectin-8: [sense primer 5′-AAGAATTCGCCGCCACCATGATGTTGTCCTTAAAC-3′ (SEQ ID NO:1), antisense primer 5′-AATCTAGACTACCAGCTCCTTACTTC-3′ (SEQ ID NO:2)].

[0273] The above described reaction mixture was subjected to an amplification program of 1 min at 94° C., 1 min at 60° C. and 2 min at 72° C. for 30 cycles.

[0274] Purification of PCR P...

example 2

Expressing Recombinant Galectin-8 Molecules in Escherichia Coli

[0283] Materials and Methods

[0284] In order to express recombinant-galectin-8 molecules (r-galectin-8 (SEQ ID NO: 4), r-galectin-8 variant1 (SEQ ID NO: 6), r-galectin-8 variant 2 (SEQ ID NO: 8)) in E. coli, the following primers were used to amplify the entire coding sequence of human galectin-8 using the cloned cDNA in pGEM vector (Promega) as a template: [0285] sense primer: 5′ACTCTAGAGCCGCCACCATGATGTTGTCCTTAAAC-3′ (SEQ ID NO:11), [0286] antisense primer: 5′-CCATATTCCTACCAGCTCCTTACTTC-3′ (SEQ ID NO:12).

[0287] In order to express recombinant-galectin-8 variant 1.1 molecule (SEQ ID NO:16) the following primers were used: [0288] sense primer: 5′CCTCTAGAATGATGTTGTCCTTAAAC-3′(SEQ ID NO: 13); [0289] antisense primer: 5′-GCCATATGCTACCAGCTCCTTACTTC-3′ (SEQ ID NO:14),

[0290] (The Xba I and Nde I restriction sites, respectively, in the above primers are underlined).

[0291] The PCR products were digested by Xba I and Nde I, ge...

example 3

Flow Cytometry Analysis of Galectin-8 Expression on Synovial Fluid Cells and PBLs and Recombinant Galectin-8 Binding to Such Cells

[0295] Materials and Methods

[0296] Peripheral white blood cells and the total cell population from synovial fluids of arthritic patients were isolated (approximately 107 cells) on a Ficoll gradient (Robbins Scientific Corporation, Sunngvale, Calif., USA) and analyzed by flow cytometry using anti-galectin-8 polyclonal antibody (generated in rabbits according to prior art protocols).

[0297] Specific binding of anti-galectin-8 was performed by the incubation of cells for 20 minutes on ice in a binding calcium buffer containing a saturating concentration of anti-galectin-8. Thereafter, a second incubation with a goat-anti-rat conjugated to fluorescein isothiocyanate (FITC) was effected. After incubation, the cells were pelleted, and analyzed in FACS analyzer (Beckton Dickinson, San Jose, Calif.). Following the initial analysis, recombinant galectin-8 (as pr...

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Abstract

Isolated polynucleotides encoding novel galectin-8 variants polypeptides are provided. Also provided are methods and kits using same for diagnosing prognosing and treating rheumatoid arthritis (RA) and other joint/chronic inflammatory diseases.

Description

[0001] This is a continuation-in-part of PCT Patent Application No. PCT / IL03 / 00960, filed Nov. 13, 2003, which claims the benefit of priority from U.S. Provisional Patent Application No. 60 / 428,691, filed Nov. 25, 2002 and U.S. Provisional Patent Application No. 60 / 426,041, filed Nov. 14, 2002, the contents of which are hereby incorporated by reference.FIELD AND BACKGROUND OF THE INVENTION [0002] The present invention relates to novel galectin sequences and use thereof in treatment or diagnosis of arthritis and other chronic inflammatory diseases. [0003] Rheumatoid Arthritis (RA) is a chronic, systemic inflammatory disease primarily involving the joints. It affects 1-3% of the population with a female to male ratio of 3:1. Constitutional symptoms may include malaise, fever and weight loss. The disease characteristically begins in the small joints of the hands and feet, usually progressing in a centripetal and symmetric fashion. Deformation of joints and disability are very common, b...

Claims

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Application Information

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IPC IPC(8): A61K38/17C07K14/705C07H21/04C12P21/06
CPCA61K38/17A61K2039/505C07H21/04G01N2800/102C07K16/18C07K16/241C07K14/4726
Inventor GOLAN, ITSHAKCASPI, DANMELNIK, LORA
Owner YISSUM RES DEV CO OF THE HEBREWUNIVERSITY OF JERUSALEM LTD
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