Chemically Synthesized SDF-1α Analogue, N33A, Is a Potent Chemotactic Agent for CXCR4/Fusin/LESTR-expressing Human Leukocytes

Abstract

Stromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1α analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1α for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1α for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1α analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1α to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS. Stromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1α analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1α for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1α for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1α analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1α to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS. Stromal cell-derived factor (SDF) 1The abbreviations used are: SDF, stromal cell-derived factor; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; Boc, t-butoxycarbonyl; CI, chemotaxis index; rh, recombinant human.1The abbreviations used are: SDF, stromal cell-derived factor; HIV-1, human immunodeficiency virus type 1; HPLC, high pressure liquid chromatography; Boc, t-butoxycarbonyl; CI, chemotaxis index; rh, recombinant human. 1 has been reported to be a primordial chemokine of the CXC subfamily and has multiple biological activities on a variety of cell types (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 2Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar, 3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). SDF-1 was initially isolated as a T lymphocyte chemoattractant and was found to be active also on monocytes but not on neutrophils (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar). However, its activity on neutrophils was subsequently shown by using Ca2+ mobilization experiments (4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). The SDF-1 gene is located on chromosome 10 (5Shirozu M. Nakano T. Inazawa J. Tashiro K. Tada H. Shinohara T. Nonjo T. Genomics. 1995; 28: 495-500Crossref PubMed Scopus (534) Google Scholar), while the genes for other known CXC chemokines are located chromosome 4 (6Oppenheim J.J. Wang J.M. Chertov O. Taub D.D. Ben-Baruch A. Tilney L.N. Strom T.B. Paul L.C. Transplantation Biology: Cellular and Molecular Aspects. Lippincott-Raven, Philadelphia1996: 187-200Google Scholar, 7Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar). SDF-1 is well conserved with only a single amino acid substitution between human and murirne molecules (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 2Nagasawa T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar). SDF-1α and SDF-1β were believed to be the result of differential splicing of a single gene, with SDF-1α missing four amino acids in the carboxyl terminus. The CXCR4/Fusin/LESTR has been identified as one of the functional receptors for SDF-1 (3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar), a member of the seven transmembrane spanning receptor superfamily (8Kelvin D.J. Michiel D.F. Johnston J.A. Lloyd A.R. Sprenger H. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1993; 54: 604-612Crossref PubMed Scopus (145) Google Scholar, 9Murphy P.M. Cytokine Growth Factor Rev. 1996; 7: 47-64Crossref PubMed Scopus (281) Google Scholar). CXCR4 was initially cloned as an orphan receptor (10Loetscher M. Geiser T. O'Reilly T. Zwahlen R. Baggiolini M. Moser B. J. Biol. Chem. 1994; 269: 232-237Abstract Full Text PDF PubMed Google Scholar, 11Federsppiel B. Melhado I.G. Duncan A.M.V. Delaney A. Schappert K. Clark-Lewis I. Jirik F.R. Genomics. 1993; 16: 707-712Crossref PubMed Scopus (162) Google Scholar, 12Nomura H. Nielsen B.W. Matsushima K. Int. Immunol. 1993; 5: 1239-1249Crossref PubMed Scopus (141) Google Scholar) and was later identified as a fusion co-factor for the entry of HIV-1 of the T lymphotropic strain (13Feng Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3624) Google Scholar). In addition to its activities on human leukocytes, SDF-1 has been shown to inhibit the fusion and replication of T lymphotropic HIV-1 in host cells bearing CD4 and CXCR4 (3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). Therefore, SDF-1 plays a pivotal role in host immune system and its defense against infection. Rapid production of cytokines and chemokines is essential for structure-function studies and design of molecules with agonist or antagonist activities. The turbocharged peptide synthesis technology (14Schnolzer M. Alewood P. Jones A Alwood D. Kent S.B.H. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (938) Google Scholar) and chemical ligation of peptide segments (15Dawson P.E. Muir T.W. Clark-Lewis I. Kent S.B.H. Science. 1994; 266: 776-779Crossref PubMed Scopus (3139) Google Scholar) permit the synthesis of chemokines in large quantity. This approach was therefore utilized to synthesize SDF-1α based on its great utility in studies of immune responses and infectious diseases. However, the thioacid peptide (SDF-1α(1–33)-αCOSH) could not be readily generated on the standard α-thiocarboxylate resin (16Canne L.E. Walker S.M. Kent S.B.H. Tetrahedron Lett. 1995; 36: 1217-1220Crossref Scopus (74) Google Scholar) due to the presence of an Asn33 residue at the ligation site in SDF-1α. Instead, alanine was coupled to the α-thiocarboxylate resin, then the mutant SDF-1α(1–33, N33A) was synthesized. In this study, we report that this SDF-1α analogue, N33A, displays full biological activity through the activation of the receptor CXCR4. This analogue, which can be rapidly produced in large quantity in the absence of contaminating peptides, will prove to be an important tool in the study of host immunity and AIDS. Human recombinant SDF-1α was purchased from PeproTech Inc. (Rocky Hill, NJ). Radioiodination of the SDF-1α and the analogue N33A was performed by a lactoperoxidase-labeling procedure. The radioactive ligands were further purified by reversed phase HPLC. The specific activity of the radioiodinated chemokines was 2200 Ci/mmol. Boc protected amino acids were obtained from the following sources: AnaSpec (San Jose, CA), Bachem (Philadelphia, PA), NovaBiochem (San Diego, CA), and Peptides International (Louisville, KY). Peptides were synthesized on a modified ABI430A instrument using in situ neutralization Boc chemistry protocols (17Canne L.E. Ferre-D'Amare A.R. Burley S.K. Kent S.B.H.J. J. Am. Chem. Soc. 1995; 117: 2998-3007Crossref Scopus (179) Google Scholar). C-terminal segments were prepared on –OCH2Pam resins (ABI, Foster City, CA). N-terminal segments were prepared on α-thiocarboxylate resin (16Canne L.E. Walker S.M. Kent S.B.H. Tetrahedron Lett. 1995; 36: 1217-1220Crossref Scopus (74) Google Scholar). Standard HF cleavage protocols were employed following N-terminal Boc removal and drying of the resin. HPLC purification was performed on Rainin HPLCs (Woburn, MA) using Vydac C4 or Dynamax C4 columns with gradient elution (A, H2O, 0.1% trifluoroacetic acid; B, acetonitrile, 0.1% trifluoroacetic acid). Electrospray mass spectrometry was performed on a Sciex API1(PE-Sciex). Ligation of the peptide segments was performed at 4 mmpeptide concentration in 6 m guanidine, 0.1 mTris, pH 7, in the presence of 33 mm thiophenol (Fluka, Switzerland) at room temperature. Ligation was monitored by HPLC and was typically complete within 24 h. Ligation was followed by HPLC purification and lyophilization. After purification, the full-length peptide was reduced at 1 mg/ml in 8 m urea (Fluka), 0.1m Tris (Fluka), 5.37 mm EDTA ((Fluka), pH 8.6, in the presence of 100 mm 2-mercaptoethanol (Fluka). Reduction occurs under a nitrogen atmosphere at 40 °C for 1 h. After complete reduction, the mixture was reconstituted with the same buffer at 0.2 mg/ml with 18.7 mm oxidized glutathione (Sigma). The solution was dispensed into a Spectrum Spectra/Por*7 dialysis membrane (Houston, TX) (M r cut-off, 3500) and the bag was placed in 1 liter of initial dialysis buffer of 8m urea, 0.1 m Tris, 1 mm EDTA, 3 mm 2-mercaptoethanol, 1.3 mm oxidized glutathione, pH 8.6. Over a period of 2 days, 4 liters of 2m urea, 0.1 m Tris, pH 8.6, was pumped into the vessel containing the dialysis bag (18Maeda Y. Ueda T. Imoto T. Protein Eng. 1996; 9: 95-100Crossref PubMed Scopus (28) Google Scholar). After lyophilization, the full-length peptide was oxidized at 1 mg/ml in 2 mguanidine HCl (Fluka), 0.1 m Tris (Fluka), pH 8.6, at room temperature in the presence of air. Folding was complete after stirring overnight and was monitored by HPLC and mass spectrometry. Human peripheral blood leukocytes were isolated from normal donors (National Institutes of Health Clinical Center Transfusion Department, Bethesda, MD) according to the established protocols in this laboratory for purification of monocytes (purity was >90%) (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar), T lymphocytes (purity, >95% CD3+) (20Xu L. Kelvin D. Ye G.Q. Taub D.D. Ben-Baruch A. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1995; 57: 335-342Crossref PubMed Scopus (65) Google Scholar) and neutrophils (purity, >98%) (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar). The chemokine receptor, CXCR4/fusin/LESTR cDNA was isolated in this laboratory and was stably transfected into human kidney embryonic epithelial 293 cells (CXCR4/293 cells) as described previously (21Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Leukocyte migration was evaluated using a 48-well microchamber (Neuroprobe, Cabin John, MD) technique as described previously (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar, 20Xu L. Kelvin D. Ye G.Q. Taub D.D. Ben-Baruch A. Oppenheim J.J. Wang J.M. J. Leukocyte Biol. 1995; 57: 335-342Crossref PubMed Scopus (65) Google Scholar,22Falk W.R. Goodwin Jr., R.H. Leonard E.J. J. Immunol. Methods. 1980; 33: 239-247Crossref PubMed Scopus (251) Google Scholar). The migration of CXCR4/293 cells was also assessed by the 48-well microchamber technique with the polycarbonate filters (10-μm pore size) (21Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) precoated with collagen type I (Collaborative Biomedical Products, Bedford, MA). The results are expressed as the the chemotaxis index (CI) representing the fold increase in the cell migration induced by stimuli versus control medium. All experiments were performed at least two times, and results from one experiment are shown. The of the between migration in response to stimuli and control was assessed by were performed using a single concentration chemokines in the presence of of ligands (19Xu L.L. MacVicar D.W. Ben-Baruch A. Kuhns D.B. Johnston J. Oppenheim J.J. Wang J.M. Eur. J. Immunol. 1995; 25: 2612-2617Crossref PubMed Scopus (76) Google Scholar, A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The binding were with a of and Bethesda, In the binding were with and and only the the obtained with native leukocytes or CXCR4/293 The of for binding by ligands was with the following in the presence of 1 in the presence of the SDF-1α analogue N33A was to and activate human peripheral blood monocytes and T that to human peripheral blood monocytes and T lymphocytes 1 with high affinity and for monocytes and T This of binding to monocytes and T cells was to that as in experiments not N33A binding to monocytes and T lymphocytes and the binding of to these cell types was also by N33A and and migration of monocytes and T cells was induced by N33A The and of N33A in the of cell migration was with that chemically synthesized N33A the and as well as response of monocytes and T cells to N33A and of N33A or were placed in the of the The cells were placed in the which were from the by polycarbonate After the filters were and the cells that the filters were The results are expressed as the representing the fold increase of cell migration in response to 2 are in with the migration response to control The of SDF-1 on neutrophils is (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar, 3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, 4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). to activity of SDF-1 for neutrophils (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google Scholar), were to Ca2+ mobilization in neutrophils at of SDF-1 (4Oberlin E. Amara A. Bachelerie F. Bessia C. Virelizier J-L. Arenzana-Seisdedos F. Schwartz O. Heard J.-M. Clark-Lewis I. Legler D.F. Loetscher M. Baggiolini M. Moser B. Nature. 1996; 382: 833-835Crossref PubMed Scopus (1478) Google Scholar). In an to the activity of SDF-1 on neutrophils, we the binding and function of N33A on neutrophils in shown in human peripheral blood neutrophils expressed a of specific binding sites for N33A 3 and native SDF-1α 3 The binding is of high affinity with of N33A and competed equally well for binding as shown by the and not also in response to N33A and that neutrophils are the cell types for SDF-1. further that N33A CXCR4 as its functional receptor, cells stably CXCR4 (CXCR4/293 cells) were type HEK 293 cells a of specific binding binding for N33A and native SDF-1α. N33A and native SDF-1 also induced a but directional migration of type cells at This is in with the that a great variety of cells CXCR4 However, cells CXCR4 expressed of specific binding sites for N33A and A N33A and competed for binding to CXCR4/293 cells and N33A and were to a directional migration of CXCR4/293 cells with and cells transfected with known chemokine and not binding or activation by N33A or these cells and in response to ligands not These results demonstrate that N33A, CXCR4 as a functional response of CXCR4/293 cells induced by N33A and CXCR4/293 cell migration was by at °C in as described previously (21Ben-Baruch A. Xu L. Young P.R. Bengali K. Oppenheim J.J. Wang J.M. J. Biol. Chem. 1995; 270: 22123-22128Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). 2 are in with the are important that in a variety of and immune as well as (6Oppenheim J.J. Wang J.M. Chertov O. Taub D.D. Ben-Baruch A. Tilney L.N. Strom T.B. Paul L.C. Transplantation Biology: Cellular and Molecular Aspects. Lippincott-Raven, Philadelphia1996: 187-200Google Scholar, 7Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar). of chemokine receptor been identified to be fusion for HIV-1 entry into host chemokines were to inhibit HIV-1 entry through of the receptors as or CXCR4/Fusin/LESTR which the fusion of or T lymphocytotropic with host cells T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar, 3Bleul C.C. Fazzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. Nature. 1996; 382: 829-833Crossref PubMed Scopus (1749) Google Scholar, Y. Broder C.C. Kennedy P.E. Berger E.A. Science. 1996; 272: 872-877Crossref PubMed Scopus (3624) Google Scholar, R. Choe S. D. M. S. Nature. 1996; PubMed Scopus Google Scholar, T. S. Y. C. 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Transplantation Biology: Cellular and Molecular Aspects. Lippincott-Raven, Philadelphia1996: 187-200Google Scholar, 7Baggiolini M. Dewald B. Moser B. Adv. Immunol. 1994; 55: 97-179Crossref PubMed Scopus (2259) Google Scholar). In the absence of blood monocytes in the a by from blood and into SDF-1 displays a that is for function in lymphocyte in of monocytes and in normal and of (1Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1274) Google K. Tada H. R. M. Nakano T. T. Science. 1993; PubMed Scopus Google Scholar, P. S.M. N. Exp. Res. 1994; PubMed Scopus Google Scholar, R. D. J. 1995; Full Text PDF PubMed Scopus Google Scholar). However, neutrophils not or these cells binding sites for and in response to SDF-1α in as further in the the role for SDF-1 as a primordial chemokine the of leukocytes further SDF-1 also has essential in T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar). SDF-1 due to and the of cell in mutant were reduced in and were reduced only in the not in the that SDF-1 is for cell and In of SDF-1 gene a T. Hirota S. Tachibana K. Takakura N. Nishikawa S. Kitamura Y. Yoshida N. Kikutani H. Kishimoto T. Nature. 1996; 382: 635-638Crossref PubMed Scopus (1999) Google Scholar). The SDF-1α analogue N33A was chemically synthesized and was shown in this study to be equally as potent as native SDF-1α in binding and human leukocytes as well as N33A has also been shown to human lymphocyte migration and to inhibit of cells by A. D. A. L. E. M. S. B. H. R. J. P. E. E. D. and E. A. The of is by chemical in which proteins can be produced or by The could a full of amino acids as well as and as or The activities of can also be by of B. 1996; PubMed Scopus Google Scholar). The of this approach is by the that functional molecules as the SDF-1α analogue N33A, a biological agonist of SDF-1 that can be utilized in studies of the immune system and host defense against AIDS. Bengali and for and and for The of this by J. Oppenheim is