Mesenchymal stem cells: biology and potential clinical uses.

There has been an increasing interest in recent years in the stromal cell system functioning in the support of hematopoiesis. The stromal cell system has been proposed to consist of marrow mesenchymal stem cells that are capable of self-renewal and differentiation into various connective tissue lineages. Recent efforts demonstrated that the multiple mesenchymal lineages can be clonally derived from a single mesenchymal stem cell, supporting the proposed paradigm. Dexter demonstrated in 1982 that an adherent stromal-like culture was able to support maintenance of hematopoietic stem as well as early B lymphopoeisis. Recent data from in vitro models demonstrating the essential role of stromal support in hematopoiesis shaped the view that cell-cell interactions in the marrow microenvironment are critical for normal hematopoietic function and differentiation. Maintenance of the hematopoietic stem cell population has been used to increase the efficiency of hematopoietic stem cell gene transfer. High-dose chemotherapy and frequently cause stromal damage with resulting hematopoietic defects. Data from preclinical transplantation studies suggested that stromal cell infusions not only prevent the occurrence of graft failure, but they have an immunomodulatory effect. Preclinical and early clinical safety studies are paving the way for further applications of mesenchymal stem cells in the field of transplantation with respect to hematopoietic support, immunoregulation, and graft facilitation.

Abnormal response to IL-5 in B-cell chronic lymphocytic leukemia.

In B-cell chronic lymphocytic leukemia, neoplastic B-lymphocytes are arrested in development. Since interleukins are essential for B-cell differentiation, we examined whether B-CLL cells were capable of responding normally to interleukins. Purified B-lymphocytes from B-CLL patients and controls were compared for their ability to proliferate and differentiate after stimulation with MCAT or SAC plus rhIL-2 or rhIL-5. When rhIL-5 was added to MCAT-stimulated cells, 8 of 10 controls showed a substantial increase in IgM production, compared with only 1 of 10 B-CLL patients. Lack of IL-5 responsiveness could provide insight into the arrested B-lymphocyte development of some B-CLL patients.

Gene transfer therapy for heritable disease: cell and expression targeting.

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Different gene transfer strategies may be required to target different tissues. Adenosine deaminase (ADA) deficiency is a good gene therapy model for targeting a rare population of pluripotent hematopoietic stem cells capable of self-renewal. We present evidence for the highly efficient gene transfer and sustained expression of human ADA in human primitive hematopoietic progenitors using retroviral supernatant with a supportive stromal layer. A stem cell-enriched (CD34+) fraction was also successfully transduced. Duchenne muscular dystrophy (DMD) is also a good model for somatic gene therapy. Two of the challenges presented by this model are the large size of the gene and the large number of target cells. Germline gene transfer and correction of the phenotype has been demonstrated in transgenic mdx mice using both a full-length and a truncated form of the dystrophin cDNA. We present here a deletion mutagenesis strategy to truncate the dystrophin cDNA such that it can be accommodated by retroviral and adenoviral vectors useful for somatic gene therapy.

Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.

The human androgen-receptor gene (HUMARA; GenBank) contains a highly polymorphic trinucleotide repeat in the first exon. We have found that the methylation of HpaII and HhaI sites less than 100 bp away from this polymorphic short tandem repeat (STR) correlates with X inactivation. The close proximity of the restriction-enzyme sites to the STR allows the development of a PCR assay that distinguishes between the maternal and paternal alleles and identifies their methylation status. The accuracy of this assay was tested on (a) DNA from hamster/human hybrid cell lines containing either an active or inactive human X chromosome; (b) DNA from normal males and females; and (c) DNA from females showing nonrandom patterns of X inactivation. Data obtained using this assay correlated substantially with those obtained using the PGK, HPRT, and M27 beta probes, which detect X inactivation patterns by Southern blot analysis. In order to demonstrate one application of this assay, we examined X inactivation patterns in the B lymphocytes of potential and obligate carriers of X-linked agammaglobulinemia.

Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells.

Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1.