Adverse reactions during stem cell infusion in children treated with autologous and allogeneic stem cell transplantation.

Adverse reactions (ARs) during the infusion of cellular therapy products (CTPs) are common in patients undergoing hematopoietic stem cell transplantation (HSCT). We retrospectively studied pediatric patients undergoing autologous and allogeneic HSCT to determine the incidence and grade of ARs during stem cell infusion and their predictors. We analyzed data from 213 patients (120 allogeneic and 93 autologous) who received at least 1 CTP, totaling 361 infusion episodes. Serious ARs, defined as grade 2 and 3, occurred in 25 and 11% of infusions, respectively. No grade 4 or 5 ARs were noted. Independent risk factors for developing a serious AR included stem cell source (PBSC vs marrow (odds ratio (OR) 1.8, 95% confidence interval (CI): 0.4-9); cord vs marrow (OR 7.3, 95% CI: 1.3-40), overall P=0.0001) but manipulated CTPs were protective (OR 0.4, 95% CI: 0.2-0.7, P=0.004). Unlike previous adult studies, WBC and granulocyte content were not found to be risk factors in this pediatric population. These data suggest that children tolerate higher WBC content during infusion of CTPs and support the use of manipulated CTP, as indicated, to reduce the risk of adverse infusion reactions.

Application of SFHR to gene therapy of monogenic disorders.

Gene therapy treatment of disease will be greatly facilitated by the identification of genetic mutations through the Human Genome Project. The specific treatment will ultimately depend on the type of mutation as different genetic lesions will require different gene therapies. For example, large rearrangements and translocations may call for complementation with vectors containing the cDNA for the wild-type (wt) gene. On the other hand, smaller lesions, such as the reversion, addition or deletion of only a few base pairs, on single genes, or monogenic disorders, lend themselves to gene targeting. The potential for one gene targeting technique, small fragment homologous replacement (SFHR) to the gene therapy treatment of sickle cell disease (SCD) is presented. Successful conversion of the wt-beta-globin locus to a SCD genotype of human lymphocytes (K562) and progenitor/stem hematopoietic cells (CD34(+) and lin-CD38-) was achieved by electroporation or microinjection small DNA fragments (SDF).

Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells.

A novel glass needle-mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34(+)/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% +/- 8.4% of CD34(+) cells and 42% +/- 14% of CD34(+)/CD38(-)cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34(+) and CD34(+)/CD38(-) cells, and retention of the ability of CD34(+)/CD38(-) cells to generate progenitor cells. Delivery of DNA constructs (currently

Micro-injection-mediated hematopoietic stem cell gene therapy.

Over the past decade, significant attention has been devoted to the development of viral vectors (i.e., retrovirus, lentivirus, adeno-associated virus) and conditions capable of transducing hematopoietic stem cells. After several years of disappointing results, recent reports in humans and other primates, most particularly the French report of successful treatment of X-linked severe combined immune deficiency (SCID) [1.], indicate that viral approaches will be successful in treating specific hematopoietic diseases. However, it is clear that alternate non-viral methods of gene delivery and genetic modification offer significant advantages, and may in fact be the only effective approach for treating certain blood diseases. In this review, we focus on glass needle-mediated micro-injection as a method for the delivery of genetic material into blood stem cells, with an emphasis on molecules capable of either compensating gene deletions/mutations or directly repairing gene mutations.

Alpha 3 beta 1 adhesion to laminin-5 and invasin: critical and differential role of integrin residues clustered at the boundary between alpha 3 N-terminal repeats 2 and 3.

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes.

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.

Monoclonal antibody recognizing a determinant on type 2 chain blood group A and B oligosaccharides detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and human colon cancer cell lines.

Altered expression of ABH blood group substances is a common feature of human colorectal carcinoma, yet it remains unclear how these structural changes influence the biological properties of tumor cells. Azoxymethane-induced rat colon tumors display many features of the human disease, thereby providing a potentially useful model to study the role of blood group substances in colon cancer progression. We have prepared monoclonal antibodies to a microsomal fraction isolated from an azoxymethane-induced rat colon tumor and selected an antibody that detects cancer-associated changes. Monoclonal antibody (mAb) 3A7 recognizes a determinant on type 2 chain blood group A (GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R) and B (Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R) oligosaccharides. Expression of the epitope detected by this antibody was developmentally regulated in rat colon, with maximal expression from day 4-21 after birth. Immunohistochemical staining and Western blotting analyses of azoxymethane-induced colon tumors revealed increased expression of the epitope in all of the 21 colonic tumors examined, including preneoplastic glands within transitional mucosa. Conventional and signet-ring adenocarcinomas that had invaded through the muscularis propria (Duke's B2) consistently showed the most intense staining with mAb 3A7, including regions depicting angioinvasion. Some of the lymph node metastases (Duke's C2) stained poorly with the antibody. The epitope was also expressed in blood group A positive human colon carcinoma cell lines, including HT29 and SW480 but not by SW620, a cell line derived from a lymph node metastasis isolated in vivo from the SW480 primary tumor, or in the blood group B cell line SW1417. The glycoproteins detected by mAb 3A7 in rat colon tumors and HT29 cells ranged in size between 50 and 200 kd, including a major species of 140 kd. Affinity chromatography of detergent lysates of normal rat colon on the blood group A specific lectin Dolichos biflorus (DBA)-agarose resulted in nearly quantitative binding of glycoprotein species detected by the antibody. By contrast, immunoreactive glycoproteins from rat colon tumors or HT29 cells bound poorly to DBA-agarose but were retained by another blood group A-binding lectin, Helix-pomatia (HPA)-agarose. These results indicate that colon carcinogenesis results in quantitative as well as qualitative changes in oligosaccharides detected by mAb 3A7 and suggest that the combined use of mAb 3A7 and blood group A-specific lectins may provide a useful tool for early detection of colon cancer.